J Plant Pbysiol. Vol. 143. pp. 520- 525 (1994)

Introduction

High-performance Liquid Chromatography with Photodiode­array Detection of Carotenoids and Carotinoid Esters in Fruits and Vegetables

PETER A BlAcs* and HUSSEIN G. DAOOD

Central Food Research Institute, Hermann Otto ut 15, H-1022, Budapest, Hungary

Received September 5,1993 . Accepted November 20,1993

Summary

A procedure for the simultaneous, one-step analysis of polar xanthophylls, xanthophyll mono- and diesters and geometric isomers of carotenes in selected fruits and vegetables using high-performance liq­uid chromatography (HPLC) and photodiode-array detector is described.

The separation of carotenoid extracts was first performed on a column of 6 /lm particles using differ­ent mobile phases and variable wavelength detectors to monitor column effluents. This procedure had limitations regarding separation efficiency and ease of spectrum analysis during the chromatographic run.

The method was further developed by using a column of 6/lm particles and a mobile phase consisting of acetonitrile-isopropanol-methanol-water (39: 52: 5: 4 v/v/v). This development permitted the isocrat­ic separation of about 45 components in less than 50 minutes. Rapid and precise identification of the indi­vidual carotenoids was achieved by using photodiode-array detector under the control of chromato­graphic software which also allowed for rapid test of peak purity and complete mapping of the whole separation profile including major lipid classes and fat-soluble materials.

A wide variety of carotenoids and carotenoid fatty acid esters were separated and quantified in selected fruits and vegetables such as spice red pepper, tomato, carrot, orange and apricot. cis isomers of {3-carot­ene, the most effective provitamin A, were also separated and differentiated from their corresponding trans forms.

Key words: Carotenoids; HPLC analysis; Photodiode array detection.

Abbreviations: DE and ME '" diesters and monoesters of carotenoids; f.wt. = fresh weight; PAD = photodiode array detection.

A carotenoid extract of biological materials is a mixture of highly colored compounds of which {3-ionone containing structure can also exhibit provitamin A activity (Bauern­feind, 1981). Recent studies have correlated the consumption of carotenoid-rich foods with reduced incidence of several types of cancer in human beings (Moon and Micozzi, 1988).

Both provitamin A active and inactive carotenoids have been found protective against cancer. Studies by Burton and Ingold (1984) showed that, at low oxygen pressure, provita­min A behaves as a good radical-trapping antioxidant and thereby retards free radical formation during the course of li­pid oxidation which has long been demonstrated to be one of the factors promoting cancer incidence. Because of the ap­parent beneficial relationship between carotenoids and some diseases, more attention is being paid to their separation and quantitation in foods by rapid, sensitive and reliable analyt­ical methods. " Correspondence.

© 1994 by Gustav Fischer Verlag, Stungan

Many analytical procedures have been proposed for the analysis of carotenoids. They included traditional color­imetry and spectrophotometry (Bauernfeind, 1981; Lich­tenthaler, 1987), chromatography (Taylor, 1985; Tee, 1991; Khachik et al., 1991) and recently super-critical fluid chro­matography (SFC) (Schmitz et al., 1989). Due to many ad­vantages, high-performance liquid (HPLC) has rapidly be­come superior to the classical analytical method which have limitations regarding reproducibility, sensitivity and ease of operation. Also, HPLC made the analysis of diverse and very susceptible carotenoid extracts easier and with minimal chemical alteration during analysis.

Diversity of carotenoid extracts from higher plants in­creases when OH-containing xanthophylls enter into acyla­tion reactions with fatty acids producing a large number of carotenoid esters differing in molecular species of fatty acid moiety. To simplify chromatograms of the HPLC separa­tion, many authors saponified carotenoid extracts to remove fatty acids (Matus et al., 1981. Chandler and Schwartz, 1987; Almela et al., 1990). On the other hand, analysis of carote­noid esters is necessary required to investigate the nature and degree of fatty acid - carotenoid esterification which have been proved to be advantageous for colour stability in raw and processed foods (Biacs et al., 1989). Therefore, separa­tion and determination of individual components of four ca­rotenoid classes: xanthophylls, monoesters, hydrocarbons and diesters by rapid and sensitive methods is of great inter­est.

In the literature reports by Philip and Chen (1988), Kha­chik and Beecher (1988), Khachik et al. (1988, 1991), Fischer and Kocis (1987) and Gregory et al. (1987) the carotenoid constituents of several fruits and vegetables have been sepa­rated and quantified. In all of these studies the method devel­opment was based on optimization of linear or step-wise gra­dient elutions using either 10 J.1m or 5 J.1m packing materials as the adsorbents of the HPLC columns.

In the present work, isocratic elution of carotenoids, ca­rotenoid esters and their geometric isomers in fruits and veg­etables, by HPLC and photodiode-array detection, is de­scribed.

Material and Methods

Carotenoid extracts were prepared by disintegrating 2 -10 grams of spice red pepper (Capsicum annuum L.), tomato (Lycopersion es­culentum L.), carrot (Daucus carota L.), orange (Citrus sinensis L.) and mandarin (Citrus reticulata L.) in a crucible mortar with quartz sand. Two grams of sodium carbonate were added to minimize acid - catalyzed isomerization of carotenoids. Following disintegration 50 mL of acetone were added and the macerate was transferred to a glass-stoppered flask and shaken for 15 min by a mechanical shaker. The macerate was filtered through a «Rundfilter MN 64 d» filter pager. The remains were shaken once again with 50 mL acetone and filtered. The two filtrate fractions were collected and the solvent was evaporated by rotatory evaporator at maximum 40°C. Ten mL of NaCl-saturated distilled water were added to the aqueous liquor remaining in the flask and the mixture was gently shaken twice with 80 mL of 1 : 1 benzene-diethyl ether.

The solvent layer was separated in a separatory funnel, washed twice with bidistilled water and dried on anhydrous sodium sulfate.

HPLC of carotenoids in fruits 521

Following evaporation of solvent (under vacuum), the pigment resi­dues were redissolved in 2 mL chloroform. The volume was brought to 10 mL with the HPLC eluent.

Thin-layer chromatography (rLG

Pigment residues were dissolved in 2 ml acetone for TLC separa­tion which was carried out on silica gel using different mobile phases according to Vinkler and Ritcher (1972), Philip (1973) and Daood et al. (1987) for red pepper, orange, and tomato and carrot extracts re­spectively.

High-performance liquid chromatography (HPLC)

A Beckman series of liquid chromatograph consisting of a Model 114 solvent delivery pump, a Model 421 controller and a Model 165 variable wavelength UV Ivisible detector. The detector signals were recorded by a Model C-R2A. Shimadzu integrator. For photodi­ode-array detection a Waters Model 990 liquid chromatograph was used. The data were stored and processed by means of NEC. APCIV power meta 2 IBM computing system. The absorption spectra of ca­rotenoids were recorded between 190 and 700 nm at the ratio of 2 spectral sec.

Separation of carotenoids was performed on columns (25 cm length x 4.6 mm i.d.) packed with lichrosorb. C-18 of either 10 ~m or 6 ~m particles. The mobile phases were: (A) acetonitrile - 15 isopropanol - water (39: 57 : 4) (B) acetonitrile - isopropanol -methanol - water (38: 52: 6: 4) Flow rate was between 0.9 and 1.5mLlmin.

Peak identification

The peaks on a chromatogram were identified by comparing their retention times and spectra with those of authentic standard prepared by TLC. Epoxide test based on HCL-Catalyzed conver­sion and shift in absorbance maxima of 5, 6 epoxide to furanoid ox­ides were used to identify the epoxide moiety of carotenoids (Bauernfeind, 1981; Baranayai et al., 1982). Identification of cis iso­mers of carotenoids was based on the appearance of extra maxima between 320 and 360 nm in the absorption spectrum of the individ­ual peaks (Chandler and Schwartz, 1987).

Quantitation of carotenoids

The carotenoids and carotenoid fatty acid esters were quantitated by comparing the areas of unknowns with those of authentic stand­ards prepared by TLC. Since zeaxanthin lutein, a-carotene, l3-carot­ene and l3-cryptoxanthin have close spectra and extinction coeffi­cients their concentrations were calculated as /3-carotene equivalent using pure standard (Sigma, USA). Concentration of each authentic standard was spectrophotometrically determined using an extinc­tion coefficient as reported by Bauernfeind (1981).

Results and Discussion

Development 0/ HPLC method

The complexity of carotenoid extracts from fruits and veg­etables as well as the variation in their polarity make it diffi­cult to separate all classes and geometric isomers of carote­noids in a single chromatographic run. For the same reason, some authors separated carotenoid classes first by open col-

522 PETER A. BIAes and HUSSEIN G. DAooD

8

9 19

6 21

II

2 16 17 23

3

5

12,50 25 50

Retention time (min)

Fig. 1: HPLC profile of mandarin peel carotenoids separated on C18, 10 ~m column. 1-17 unesterified xanthophylls, 8-14 mono­esters of dihydroxy xanthophylls, 15-18 not well identified, 19-23 esters of /3-cryptoxanthin, 24-26 lutein and zeaxanthin diesters.

umn (OCC) or TLC prior to liquid chromatography (Philip and Chen, 1988; Khachik et al., 1989).

There are however, several possibilities to increase separa­tion efficiency of a reversed-phase column under the condi­tions of isocratic elution. Factors, most likely to affect effi­ciency of the column may include diameter of parking mate­rial and composition of the mobile phase. Fig. 1 shows the separation profile of mandarin peel carotenoids on a 10 ~m C18 column eluted with mobile phase (A). Unlike in ad­sorption chromatography on silica plates, the individual es­ters of the same carotenoid, differing only in two acyl car­bons, were separated in partition chromatography on revers­ed-phase column. Free xanthophylls and monoester of di­hydroxy carotenoids eluted before 14 min and diester of di­hydroxy carotenoids as well as monoesters of monohydroxy carotenoids eluted between 14 and 45 min on the chromato­grams. Peak purity displacs provided by the chromagraphic software indicated high degree of peak impurity for most of the separated carotenoids. The interference between two or more constituents have influenced both identification and quantitation of carotenoids.

HPLC of complex carotenoid extracts was further devel­oped by using packing material of 6 ~m particles and replac­ing, in part, isopropanol with methanol in the mobile phase. With the newly elaborated system, free xanthophylls, mono­esters and hydrocarbon carotenes appeared with a retention time of 18 min and carotenoid diesters having long chain fatty acids required more than 50 min to completely elute from the column. In order to reduce their retention time and to have narrower peaks the flow rate was increased from 0.9 to 1.5 mL after 18 min (Fig. 2). A total of 37 components could be separated from mandarin or orange peel in less than 40 min. In the case of more complex extracts such as that of

red pepper the pigment was fractionated into components in less than 50 min. Similar elution profile of red pepper carote­noids have been obtained by a stepwise or linear gradient elution (Gregory et al., 1989; Fisher and Kocis, 1987).

The recently elaborated system also provided a good sepa­ration of geometric isomers present even in diverse plant ex­tracts such as orange peel. Mono- and polycis isomers of {3-carotene, capsanthin diester violaxanthin diesters and other carotenoids could be separated from their all-trans geometric isomers of different carotenoids. In specific studies Matus and coworkers (1981) and Quackenbush and Smallidge (1986) sed gradient mobile phase systems to separate geomet­ric isomers of xanthophylls and {3-carotene respectively. Isocratic elution of such isomers has also been reported, to be performed on normal phase lime columns to separate in­dividuals of saponified plant extracts (Tsukida et al., 1982; Chandler and Schwartz, 1987; Almela et al., 1990). Fig. 3 shows the HPLC chromatograms of carotenoid extracts from fresh and canned carrot. Canning of carrot at 121°C (autoclaving) for 15 min resulted in a rapid conversion of all­trans carotenes (mainly (3-carotene) to cis structures. In addi­tion, many derivative having higher polarity appeared in a shorter retentition time one chromatogram of canned carrot extract.

Photodiode-array detection

Photodiode-array detector (PAD) has rapidly become a powerful tool in the qualitative analysis of both simple and complex biological materials. The high monitoring ability of the detector in the UV and visible range of spectrum allows for the simultaneous detection of lipid and lipid-soluble ma­terials. As a result, three groups of lipophilics can appear on

9 II 13 15

Orange peel

2 7 21

IG

5 22 25

28

I 3

32 33

, , , 10 20 30 40

Retention time (min)

Fig.2: HPLC profile of carotenoids from orange peel. Separation was carried out on C-18, 6~m column. Detection was at 440nm. For identification see Table3.

3

2 2

Fresh Cann~d

5

_ ..... , ----~---__t,- _ 1-1 ----+---------11-

~O 10 20~0 10 20

III R~t~ntlon tlm~(mln) III R~t~ntlon tlm~ (min)

Fig. 3: HPLC separation of carotenoids extracted from fresh and canned carrot using C-18 column of 6 11m particles. 1: lutein, 2: a­carotene, 3: ,B-carotene, 4: mono-cis ,B-carotene, 5: polycis {3-carot­ene.

the screen: polar lipids, colorless carotenoids and colored ca­rotenoids.

One of the important displays provided by PAD-manager software is the contour map. This type of analysis assisted to detect polar lipids as the first column effluents followed by the less polar species except triglycerides which are strongly retained on the column due to their high partition coeffi­cient in the C-18 phase. Colorless carotenes such phytoene and phytofluence as well as vitamin A, if present, can easily be monitored and distinguished according to their absorb­ance in the range between 250 nm and 380 nm. By a series of desaturation reactions colored carotenes, which have more chromophores in their molecule than phytoene, are formed. They absorb intensively in the visible range of spectrum and appear yellow orange or red in color.

Exploiting such a display, selection of the best single moni­toring wavelength or dual-wavelength monitoring program became attainable and easy to apply.

A better analysis of the spectrum for the individual peaks appeared on a chromatogram was achieved by the perspec­tive three dimensional presentation of absorbance as a func­tion of time and wavelength. The new PADs with recent chromatographic softwares provided 3-D plots from differ­ent angles and, thereby, enabled to detect even hidden or overlapped peaks.

Carotenoid content of selected fruits and vegetables

Carotenoids from spice red pepper, tomato and orange peels were qualitatively and quantitatively analyzed by this method. Capsanthin, capsorubin, violaxanthin and t1-carot­ene were predominant in red pepper fruit (Table 1). Crypto­capsin, which had been reported by Almela et al. (1990) to be a major component in red pepper, was found in low con­centration in the sample analyzed in this work. Qualitative

HPLC of carotenoids in fruits 523

Table 1: Carotenoid composition and content of two fresh spice red pepper varieties as estimated by HPLC.

Carotenoid Spanish Hungarian Negral Mihalyteleki

Ilg/g f.wt.

Neoxanthin 1.32 1.42 Capsorubin 2.23 1.73 Violaxanthin 13.12 10.20 Capsanthin 8.30 25.30 Antheroxanthin 3.42 6.90 Mutatoxanthin 5.23 5.49 Zeaxanthin 7.30 12.87 Neolutein 5.57 1.94 {3-cryptoxanth 5.43 6.87 8-apo-carotenal 15.08 4.72

Monoesters (ME) Capsorubin ME-l 7.75 10.39 Capsorubin ME-2 14.20 23.30 Violaxanthin ME 26.40 16.51 Capsanthin ME-I 14.97 24.10 Capsanthin ME-2 26.95 44.38 Cryptocapsin ME-2 18.13 9.52 Auroxanthin ME 13.89 23.89 Antheroxanthin ME 22.90 33.93 Cryptoflavine ME 4.64 5.0 {3-cryptoxanthin ME 9.77 5.72 Cryptocapsin ME-l 4.25 2.70

{3-carotene epoxide 5.89 14.98 .B-carotene (trans) 71.49 63.63 .B-carotene (9- and IS-cis) 9.90 23.02 Polycis {3-carotene 6.70 5.69

Diesters (DE) Capsorubin DE-l 3.34 4.43 Cis-capsorubin DE 3.01 1.72 Capsorubin DE-2 10.77 4.81 Capsanthin DE-l 7.54 14.31 Violaxanthin DE-l 11.65 2.64 Capsanthin DE-2 23 .67 28.45 Capsorubin DE-3 12.28 6.96 Antheroxanthin DE 4.81 2.59 Mutatoxanthin DE 7.26 2.97 Capsanthin DE-3 24.51 19.81 Cis-mutatoxanthin DE 4.32 4.15 Polycis capsanthin DE 4.53 0.75 Lutein DE 2.18 1.45 Zeaxanthin DE-l 7.61 5.56 Zeaxanthin DE-2 4.46 7.50

and quantitative data on the individual free xanthophylls, monoesters carotenes, diesters as well as their derivatives and isomers in red pepper fruits are available for the first time here. Quantitation of the major carotenoids and carotenoid esters of red pepper have been achieved by Gregory et al. (1987), but he couldn't identify many peaks on the HPLC chromatogram.

In tomato fruit (Table 2) lycopene, t1-carotene, lycoxan­thin and lutein were abundant with lycopene being the major component. Geometric isomers and other cartenoids could be estimated as mimor constituents of tomato pig­ments. Although provitamin A carotenoids are present in

524 PETEll A. BIACS and HUSSEIN G. DAOOD

small amounts in tomato, they attract an increasing atten­tion since this vegetable is consumed in a considerable amount daily and thereby, can compensate dietary vitamin A. Presence of cis isomers of the major pigments may reflect the possible function of carotenoids as accessory com­ponents in the metabolism of light energy in chloro-or chro­moplasts.

HPLC analysis of carotenoids from orange peels are first reported in this article (Table 3). The major pigments of orange peel were identified as neoxanthin, citraurenin cit­raurin and 8-apo-carotenol, whereas mutatoxanthin anthero­xanthin, ~-carotene, ~-citraurol and ~-cryptoxanthin were the minor constituents. ~-citruarinene ester, the pigment re­sponsible for the reddish or deep orange color in oranges, eluted with retention time very close to that of a-carotene. Spectral characteristics as manipulated by the chromato­graphic software as well as disappearance of the peak on chromatographs of saponified extracts confirmed the ab­sence of a and 'Y-carotene. As regards nutritional value, one gram of orange peel can supply 0.72-6.75 Jig provitamin A (0.75-6.251U). In the case of mandarin, the peel distributed mainly ~-cryptoxanthin, violaxanthin neoxanthin and muta­toxanthin esters. ~- and a-carotene and lutein diesters were also present as minor components. One gram of mandarin peel gave 17.7 -18.3 Jig provitamin which is equal to 16.4-17.1IU revealing the high nutritional value of such a by­products.

Conclusion

The newly developed, HPLC method manifested its im­portance in the simultaneous separation of a wide variety of carotenoids including the physiologically most important xanthophylls (neoxanthin, violaxanthin, lutein, zeaxanthin etc.), carotenes, and their geometrical isomers. High effi­ciency, sensitivity and reliability of this method possible its application for on-line analysis of different biological mate-

Table 2: Carotenoid composition and content of tomato as esti­mated by HPLC.

Carotenoid

Neoxanthin Violaxanthin Antheroxanthin Mutatoxanthin Lutein Neolutein (j-cryptoxanthin Lycoxanthin Neurosporin Lycopene Neolycopene 'Y-carotene a-carotene (j-carotene monocis-(j-carotene polycis-i3-carotene

Concentration ILg/g f.wt.

0.15 0.27 0.19 0.17 0.60 0.17 0.10 0.78 0.07

38.34 0.18 0.04 0.08 1.41 0.07 0.11

Table 3: Carotenoids composition and content of Citrus fruit peel as estimated by HPLC.

Carotenoid Mandarine Orange 1Lg/ g outer layer

Neoxanthin 0.07 (I)'" trace Violaxanthin 0.25 (2) 1.86 (2)'" Luteoxanthin 0.26 (3) 0.32 (3) (j-citraurin trace 0.19 (4) Zeaxanthin 0.58 (4) 0.96 (5) Neolutein 0.50 (6) /3-cryptoxanthin 0.56 (5) 1.48 (7) (j-citraurinene 0.57 (6) 1.82 (8)

Monoesters (ME)

Neoxanthin ME-I 4.70 (8) 5.01 (9) Neoxanthin ME-2 2.47 (9) 1.31 (10) Auroxanthin ME 3.55 (10) 8.12 (11) Luteoxanthin ME 2.57 (11) Violaxanthin ME-I 6.16 (12) Violaxanthin ME-2 2.77 (12) 10.51 (13) Antheroxanthin ME 1.96 (13) J3-citraurin ME-I 3.55 (14) J3-citraurin ME-2 0.44 (14) 8.12 (15) (j-citraurinene ME 1.65 (16) 0.93 (17) /3-cryptoxanthin epoxide ME 0.89 (18) 1.00 (20) (j-cryptoxanthin ME-I 7.49 (19) 1.55 (22) (j-cryptoxanthin ME-2 4.78 (22) 2.40 (25) (j-cryptoxanthin ME-3 3.52 (25) 2.10 (28)

(j-carotene 2.00 (15) 0.77 (16)

Diesters (DE) /3-citraurol DE-l 1.19 (18) Mutatoxanthin DE 2.70 (17) 0.70 (21) J3-citraurol DE-2 2.53 (23) Neoxanthin DE-l 1.19 (20) 0.54 (23) Violaxanthin DE-l 1.42 (21) 1.13 (24) Antheroxanthin DE 0.81 (23) 0.57 (26) Violaxanthin DE-2 0.93 (24) 0.85 (27) Lutein DE-l 0.19 (29) Lutein DE-2 0.53 (26) 0.46 (29)

Zeaxanthin DE-l 0.07 (27) 0.96 (30) Zeaxanthin DE-2 0.32 (28) 1.30 (31)

... Numbers between brackets are for identification on Fig. 2.

rials. The use of a modern, PAD detector under the control of chromatographic software manifolds the utility of the method by allowing simultaneous detection of lipid com­ponents during analysis of carotenoids.

References

AumA, 1., T. M. L6PEZ-ROCA, M. E. CANDElA, and M. D. ALd.­ZAR: Separation and determination of individual carotenoids in a Capsicum cultivars by normal-phase high performance liquid chromatography. J. Chromatogr. 502, 95-106 (1990).

BAllANYAl, M., Z. MATUS, and J. SZABOLCS: Determination, by HPLC, of carotenoids in paprika products. Acta Aliment. 11, 309-323 (1982).

BAUERNFEIND, J. CH.: Carotenoids as colorants and vitamin A pre­cursors. Academic Press, New York (1981).

BIACS, P., H. G. DAOOD, A. PAVISA, and F. HA.JnD: Studies on the ca­rotenoid pigments of paprika (Capsicum annuum L. var. SZ-20). J. Agric. Food Chern. 37, 350-353 (1989).

BURTON, G . WAND, and K. V. INGOLD: ~-carotene: An unusual type of lipid antioxidant. Science 224, 569-573 (1984).

CHANDLER, L. A. and S. J. SCHWARTZ: HPLC separation of cis-trans carotene isomers in fresh and processes fruits and vegetables. J. Food Sci. 52, 669-672 (1987).

DAOOD, H. G., P. BIACS, A. HOSCHKE, M. VINKLER, and F. HAJDU: Separation of tomato fruit pigment by TLC and HPLC. Acta Aliment 16, 339-350 (1987).

FISHER, C. and J. A: Kocis: Separation of paprika pigments by HPLC. J. Agric. Food Chern. 35,35-57 (1987).

GREGORY, G. K., T. S. CHEN, and T. PHIUP: Quantitative analysis of carotenoids and carotenoids esters in fruits by HPLC: Red bell peppers. J. Food Sci. 52, 1071-1073 (1987).

KHACHIK, F., G. R. BEECHER, and W. R. LUSBY: Separation, identifi­cation and quantification of the major carotenoids in extracts of apricots, peaches, cantaloupe and pink grapefruit by liquid chro­matography. J. Agric. Food Chern. 37,1465-1473 (1989).

KHACHIK, F., G. R. BEECHER, and B. G. MUDLAGIRI: Separation, identification and quantitation of carotenoids in fruits, vegeta­bles and human plasma by high performance liquid chromato­graphy. Pure App!. Chern. 63, 71-80 (1991).

KHACHIK, F., G. R. BEECHER, and N. F. WHITTAKER: Separation, identification of carotenoids and carotenol fatty esters in some squash products by liquid chromatography. 2. Isolation and Characterization of carotenoids and related esters. J. Agric. Food Chern. 36,938 -946 (1988).

LICHTENTHALER, H. K.: Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology 148, 350-382 (1987).

HPLC of carotenoids in fruits 525

MATUS, Z., M. BARANYAI, G. T6TH, and J. SZABOLCS: Identifica­tion of oxo, epoxy and som cis-carotenoids in high perform­ance liquid chromatography. Chromatographia 14, 337-340 (1981).

MOON, T. E. and M. S. MICOZZI: Nutrition and cancer prevention: Investigating the role of micronutrients; Marcel Dekker: New York, Basel, 1988.

PHILIP, T.: The nature of carotenoid esterification in Citrus fruits. J. Agric. Food Chern. 6, 964-966 (1973).

PHILIP, T. and T. S: Chen: Development of a method for the quan­titative determination of provitamin a carotenoids in some fruits. J. Food Sci. 53, 1703-1706 (1988).

QUACKENBUSH, F. W. and R. L. SMALUDGE: Nonaqueous reverse phase liquid chromatographic systems for separation and quan­titation of provitamin A. J. Assoc. Off. Ana!. Chern. 69, 767-772-772 (1986).

SALEH, M. and B. TAN: Separation and identification of cis/trans ca­rotenoid isomers. J. Agric. Food. 39, 1438-1448 (1991).

SCHMITZ, H. H., W. E. ARTZ, C. L. POOR, J. M. DIETZ, and J. W. ERDMAN, Jr.: High performance liquid chromatography and ca­pillary supercritical-fluid chromatography separation of veget­able carotenoids and carotenoid isomers. J. Chromatogr. 479, 261-268 (1989).

TAYLOR, R. F.: Chromatography of carotenoids and retinoids. Adv. Chromatogr.22, 157 -213 (1985).

TEE, E. S. and C. L. LIM: Analysis of carotenoids and retinoids: A re­view. Food Chern. 41, 147 -193 (1991).

TSUKIDA, K., K. SAIKI, T. TAKII, and Y. KOYAMA: Separation and de­termination of cis/trans j1-carotene by HPLC. J. Chromatogr. 245,359-364 (1982).