Practical Guide to Establishing Palm Carotenoids Profiles ... ? 13 Practical Guide to Establishing

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13Practical Guide to Establishing Palm Carotenoids Profiles by HPLC with Three Dimensional Diode Array DetectorBonnie Tay Yen Ping* and Choo Yuen May*CINTRODUCTIONrude palm oil is one of the richestplant source of carotenoids in termsof retinol (pro-vitamin A) equiva-lents. Carotenoids are naturallyoccurring C-40 plant pigments. Their con-jugated polyenic chromophore is responsiblefor the characteristic light absorbing pro-perties. Hydrocarbon and oxygenated caro-tenoids are termed carotene and xantho-phyll respectively. Carotene, in particular-carotene and -carotene have long beenassociated with pro-vitamin A activity. Otherminor carotene such as -carotene and-zeacarotene also possess vitamin A activ-ity (Choo, 1995).Aside from pro-vitamin A activity, caro-tene such as -carotene, -carotene andlycopene are effective antioxidant and sing-let oxygen quenchers (Dimascio et al., 1989).Serbinova et al. (1992) found that the orderof inhibition by palm-based carotene in invitro lipid peroxidation was -carotene >lycopene > -carotene. There are consider-able evidence from epidemiological and ani-mal data linking -carotene and other caro-tene to decrease risk of some cancers (Ziegler,1989; Ziegler et al., 1996a,b). Another inter-esting finding is that -carotene is 10 timesmore potent as an anti-cancer agent than-carotene (Murakoshi et al., 1989; 1992).Practical Guide to Establish-ing Palm CarotenoidsProfiles by HPLC with ThreeDimensional Diode ArrayDetectorManorama et al. (1993) showed that redpalm oil (RedPO) was more effective inpreventing chemical carcinogenesis in ratsin comparison to refined bleached deodor-ized palm olein (RBDPOo), and attributedthis effect to the carotenoids present in theformer. The in vivo and in vitro chemicalcarcinogenesis studies by Tan and Chu(1991) in the rat hepatic cytochrome-P450-mediated monoxygenase system showed theorder of anti-tumour reactivity was palmoil (with carotenoids) > -carotene > cantha-xanthin > palm oil (without carotenoids).The various physiological potential ofpalm carotenoids warrants identification ofthe carotenoids present in palm oil. Thequantification of carotene content by thePORIM Test Method using UV-Visible spec-trophotometer, sums all the carotene as-carotene. The use of high performanceliquid chromatography (HPLC) is very use-ful in the qualitative identification of palmcarotenoids because it allows the display ofthe various carotenes in the chromatograms.This paper describes a step by step guideto analyse crude palm oil carotenoids usinga HPLC-three dimensional photodiode arraydetector (PDA).PREPARATION OF SAMPLESThe oil sample was saponified according toPORIM Test Method, but with slight changes.About 5 g of oil and 5 ml of 50% ethanolicKOH were heated at 50C in a water bath* Malaysian Palm Oil Board,P.O. Box 10620,50720 Kuala Lumpur, Malaysia.14Palm Oil Developments 33under a stream of nitrogen for 30 min. Thesaponified sample was then cooled to roomtemperature and extracted with 50 mlportions of petroleum ether until the super-natant became colourless. The pooled petro-leum ether extracts were washed four timeswith 50 ml portions of distilled water anddried over anhydrous sodium sulphate. Thenthe extract was dried in a rotary evaporatorat 50C. The dried extract was dissolved ina known volume of mobile phase containingan antioxidant before it was injected into theHPLC.HPLC CONDITIONSThe carotene profiles analyses was perform-ed using a HPLC with Waters 990 seriesPDA. The PDA was set at spectral range of222-800 nm. The isocratic non-aqueous sepa-ration system developed by Yap et al. (1991)was used. The analyses used a Metaphasereverse phase C18 column (4.6 mm i.d. x25 cm, stainless steel, 5 m spherical par-ticles), and the solvent system was aceto-nitrile:dichloromethane (89:11 v/v) at a flowrate of 1.0 ml min-1.ESTABLISHING CAROTENE PROFILEUSING THREE DIMENSIONAL PDAA concentrated extract was prepared bysaponification of the crude palm oil priorinjection as described. The extract wasdissolved in small amount of mobile phaseand injected into the HPLC-PDA system.The wavelength at 286 was chosen to monitorthe profile. After analysis, the differentchromatograms at the wavelength maximaof the various carotene were retrieved fromthe scanned data file. Figure 1 shows theHPLC chromatogram at 444 nm. Figure 2shows the three dimensional (3D) chroma-togram at 444 nm, where the UV spectraand chromatogram can be monitored simul-taneously (Figure 2). The 3D chromatogramhere shows that the peaks designated for-carotene and -carotene is not properlyresolved using the chromatographic condi-tions. There are several spectra hiddenbehind the -carotene and -carotene peaksrespectively, which may be the isomers ofthese carotenes.Table 1 lists the chromatographic reten-tion time and absorbance maxima of thecarotenes obtained from the PDA detector.Identification was done by comparison withthe published spectral data.Peak area normalization was used in thiscase for quantification. Table 2 shows thecrude palm oil carotene profiles expressedas percentage composition. The peak areais based on the peak from the caroteneabsorbance maxima. Quantification usingexternal or internal standard calibration isFigure 1. Crude palm oil carotenoids scanned at 444 nm.The peak numbers here are the same as those in Table 1.Note: phytoene and phytofluene are not shown in this chromatogrambecause they are detected only at 286 and 347 nm respectively.0 20 40 60 Time (min)1 3548910a210b6715Practical Guide to Establishing Palm Carotenoids Profiles by HPLC with Three Dimensional Diode Array DetectorFigure 2. A 3D chromatogram of palm carotenoids.TABLE 1. CRUDE PALM OIL CAROTENOIDS ORDER OFELUTION AND ABSORBANCE MAXIMA Peak Retention Carotenes Absorbance maxima (nm) time This study Davies, 19761 29.1 Lycopene 444 468 500 448 473 5042 31.5 -Zeacarotene 400 423 448 398 421 4493 34.0 -Zeacarotene 400 430 449 407 427 4544 35.0 Neurosporene 415 440 462 416 440 4705 38.8 -Carotene 425 459 487 428 458 4906 40.0 -Carotene 436 461 488 437 462 4927 41.8 -Carotene 381 402 426 380 401 4268 46.2 cis--Carotene 333 415 442 468 -9 49.2 -Carotene 420 445 474 420 442 47210a 53.2 -Carotene 430 452 477 425 450 47710b 58.5 cis--Carotene 330 420 440 480 -11 64.3 Phytoene 275 287 298 276 286 29712 57.8 Phytofluene 332 345 362 331 347 366TABLE 2. CAROTENE COMPOSI-TION (%) BASED ON PEAK AREACarotenes %Lycopene 0.9-Zeacarotene 0.2-Zeacarotene 1.1Neurosporene 0.3-Carotene 1.2-Carotene 1.2-Carotene 0.2cis--Carotene 6.2-Carotene 39-Carotene 48cis--Carotene 0.6Phytoene 0.03Phytofluene 0.2not practical for analysis using the HPLC-PDA method. This is because the purecarotene standard may degrade during thelong separation time of 65 min. The insolu-bility of the carotene standard in the mobilephase may also affect quantification.VARIABLE WAVELENGTH UV-VISIBLEDETECTOR (VWUV-Vis) DETECTORvs. PDAYap et al. (1991) were the first researchersto report on the method to identify palmcarotenoids using HPLC with a VW UV-Visdetector. However, there are certain limita--carotene-carotene1(AU)0400600800 0 20 40 60Time (min)Wavelength16Palm Oil Developments 33tions to this method. The VW UV-Vis de-tector only allows any wavelength to bemonitored sequentially. Palm carotenoidsabsorb at different wavelength maxima, i.e.phytoene (286), phytofluene (347), lycopene(472), -zeacarotene (421), -zeacarotene(427), neurosporene (440), -carotene (456),-carotene (462), -carotene (400), cis--carotene (444), -carotene (444) and -caro-tene (453). Up to 11 time injections is neededduring analysis of a sample. The analysistime is usually long at about 80 min.Lycopene, -carotene and -carotene wereidentified using the Sigma standards. Forthe other minor palm carotene where stand-ards are unavailable, such as -zeacarotene,-zeacarotene, phytoene, phytofluene, -caro-tene, -carotene and -carotene, they werecollected individually. Their spectra wasthen recorded on a UV-Visible spectropho-tometer. This method is cumbersome andnot practical for routine analysis of palmcarotenoids. Besides, the peaks have to bewell resolved prior collection.The PDA offers an easier and quicker wayto identify carotenoids qualitatively. This isbecause, unlike the VW-UV Vis detector, thesample needs to be injected once only at anychosen wavelength. The chromatograms atmultiple wavelengths can be retrieved fromthe data files after analysis. The PDA alsoallows all the carotenes to be identified bytheir elution sequence and absorption spec-tra. The PDA is able to accumulate spectraldata rapidly without disturbing the chroma-tographic process. After analysis, the spec-trum of the individual carotene peak isextracted from the spectral data and com-pared with the spectra of carotenes inliterature. Identification of carotenes can beachieved tentatively based on their charac-teristic spectral maxima and shape. How-ever, to identify the structure absolutely,other methods such as MS , NMR and IRare required.CONCLUSIONThe analyses of palm carotenoids by threedimensional HPLC-PDA was found to bemore convenient for routine analyses ofcarotene profiles compared to a VW-UV Visdetector. No multiple injections of a samplewere needed and this saved time. Further-more, the 3D chromatogram enables theunresolved peaks to be detected. The diodearray detector accumulates the spectra whichcan be retrieved after the analysis makingidentification easier. The minor carotenescan be tentatively identified even thoughno commercial standards are available.Therefore, the HPLC-PDA can be used asa routine method to qualitatively identifypalm carotenoids.ACKNOWLEDGEMENTThe authors thank the Director-General ofMPOB for permission to publish this paperand Keck Seng Sdn. Bhd. for supplying thecrude palm oil samples. Thanks are also dueto the supporting staff of Processing Re-search Group for their technical assistance.REFERENCESCHOO, Y M (1995). Carotenoids from palmoil. Palm Oil Developments No. 22:1-5.DAVIES, B H (1976). Carotenoids. Chemis-try and Biochemistry of Plant Pigments(Goodwin, T W ed.). Academic Press, Lon-don. Vol. 2.DIMASCIO, P; KAISER, S and SIES, H(1989). Lycopene as the most efficient bio-logical carotenoid singlet oxygen quencher.Arch. Biochem. and Biophys., 274:532-538.MANORAMA, R; CHINNASAMY, N andRUKMINI, C (1993). Effect of red palm oilon some hepatic drug-metabolizing enzymesin rats. Food and Chem. Toxicology, 31: 583-588.MURAKOSHI, M; TAKAYASU, J; KIMURA,O; KOHMURA, E; NISHINO, H; IWA-SHIMA, A; OKUZUMI, J; SAKAI, T; SUGI-MOTO, T; IMANISHI, J and IWASAKI, R(1989). Inhibitory effects of -carotene onproliferation of the human neuroblastoma17Practical Guide to Establishing Palm Carotenoids Profiles by HPLC with Three Dimensional Diode Array Detectorcell line GOTO. J. of the National CancerInstitute, 81 (21):MURAKOSHI, M; NISHINO, H; SATOMI,Y; TAKAYESU, J; HASEGAWA, T andTOKUDA, H (1992). Potent preventiveaction against carcinogenesis spontaneousliver carcinogenesis and promoting stage oflung and skin carcinogenic in mice are sup-pressed more effectively by -carotene than-carotene. Cancer Research Baltimore,52:23,6583-6587.SERBINOVA, E; CHOO, Y M and PACKER,L (1992). Distribution and antioxidantactivity of a palm oil carotene fraction inrats. Biochem. Internat., 28: 881-886.TAN, B and CHU, F L (1991). Effects ofpalm carotenoids in rat hepatic cytochromeP450-mediated benzo(a)pyrene metabolism.Amer. Journal of Clinical Nutrition, 53:1071S-1075S.YAP, S C; CHOO, Y M; OOI, C K; ONG,A S H and GOH, S H (1991). Quantitativeanalysis of carotenes in the oil from differentpalm species. Elaeis Vol. 2 No.2: 369-378.ZIEGLER, R G (1989). A review of epide-miologic evidence that carotenoids reducethe risk of cancer. J. of the National CancerInstitute, 119: 116-122.ZIEGLER, R G; COLAVITO, E A; HARTGE,P; MCADAMS, M J; SCHOENBERG, J B;MASON, T J and FRAUMENI, J F (1996a).Importance of -carotene, -carotene andother phytochemicals in the etiology of lungcancer. J. of the National Cancer Institute,88: 612-615.ZIEGLER, R G; MAYNE, S T andSWANSON, C A (1996b). Nutrition andlung cancer. Cancer Causes Control, 7: 157-177.


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