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DEVELOPMENT OF AN HPLC/DAD METHOD FOR DETERMINATION OF PHENOLlC
PROFILE lN PORTUGUESE OLlVE FRUITS
Ana F. Vinha', Paula B. Andrade', Branca M. Silva', José A. Pereira', Patricia Valentão', Rosa M. Seabra',
M. Beatriz Oliveira'
CEQUPI 'Serviço de Farmacognosia and 'Serviço de Bromatologia, Faculdade de Farmácia, Universidade
do Porto, R. Anibal Cunha, 164, 4050-047 Porto. 'Escola Superior Agrária, Instituto Politécnico de Bragança,
Quinta de Sta. Apolónia, Apartado 172, 5300 Bragança.
INTRODUCTION
Polyphenolic compounds inliuence the sensorial properties 01 olive fruits and virgin olive oils and are imporlant
markers for studying fruit characteristics of different cultivars and for controlling oil production processes (Romani
el aI., 1999) .
A few chromatographic methods have been used to study the phenolic compounds of olive fruit (Soler-Riv~s el
ai., 2000; Capasso el ai., 1992; Ficarra el ai., 1991; de Laurentis el aI., 1997; Baracco et aI., 1995). This
communication reports the development of a new HPLCIDAD methodology to separate, identify aml quanlily lhe
phenolic compounds from Portuguese olive fruit cultivars (Cobrançosa, Madura/and Verdea~.
EXPERIMENTAL
Extraction 01 Phenolic Compounds Irom Olive Fruits. Each olive fruit sample (ca. 1.5 g) was thoroughly
mixed with methanol until complete extraction of the phenolic compounds (negative reaclion to NaOH 20%) The
methanolic extract was filtered, concentrated to dryness under reduced pressure (40ºC) and redissolved in acid
water (pH 2 with HCI) (= 50 mL). Tlla aqueous solution was tllen passed tllrough an ISOLUTE C18 (NEC)
column, previously conditioned with 60 mL of methanol and 140 mL of acid water (pH 2 with HCI). Tlle loaded
cartridge was washed with n-hexane and phenolic compounds were eluled wilh methanol. Tlle mp.thAnolic
extract was evaporated to dryness under reduced pressure (40ºC), redissolved in metllano l (4 mL) and 20 JlL
were analysed by HPLC.
HPLC Analysis 01 Phenolic Compounds. Separation of phenolics was achieved with an analytical HPLC L1nit
(Gilson), using a Spherisorb ODS2 (25 .0 x 0.46 cm;
5~lm,
partiele size) column. The solvent system used
WilS
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-
gradient of water-formic acid (19:1) (A) and methanol (B): O' - 5% B, 3' - 15% B, 13' - 25% B, 25' - 30% 8,
35% 8, 39' - 40% 8, 42' - 45% 8, 45' - 45% 8, 50' - 47% 8, 60' - 48% 8 , 64' - 50% B, 66' - 100% 8. Tlle solvent
flow rate used was 0.9 mUmin. Detection was achiaved with a Gilson diode array detector (DAD), ilnd
chromatograms were recorded at 280 and 320 nm. Phenolic compounds quantification was achieved by Ille
absorbance recorded in the chromatograms rela tive to externa I standards.
RESULTS AND DlSCUTION
The results demonstrale that the Cobrançosa cultivar has the same qualitative composilion as MaduraI, being
charactensed by the presence of nine identified compounds: hydroxytyrosol, 5-O-caffeoylquinic acid, verbascoside,
luteolin 7-O-glucoside, oleuropein, rutin, apigenin 7-Q-glucoside, quercetin 3-Q-rllamnoside and luteolin (Figure
1) . Verdeal cultivar exhibited a similar phenolic composition, but verbascoside was not presen!.
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Figure 1. HPLC pi1enolic profile of a olive fruit sample. (1) hydroxytyrosol; (2) 5-O-caffeoylquinic: (3)
verbascoside; (4) luteolin 7-0-glucoside; (5) oleuropein; (6) rutin; (7) apigenin 7-O-glucoside; (8) quercetin 3O-rhamnoside; (9) luteolin.
ACKNOWLEGMENTS
Branca M. Silva is grateful to Fundação para a Ciência e a Tecnologia for a grant (PRAXIS XXI/BD/21339/991.
REFERENcES
A Romani. N. Mulinacci P. Pinelll F E Vinderi A, Cima!o
C. Soler-Rivas
J C. Esmo.. H. Wichers J Sei. Eood Aqric.
J. Agric. Food Chem. 47 (1999) 964-967,
ao (2000),
1013-1023.
CapaSSQ A. EvirJente A, SCQgnamlgllo F. PhY!Qchem Anal 3 (1992) 270·275
P Fjcarra A F;carra A de PasQuale M T Monforte M L Celebro FarmacQ46 (1991) 803-815
N de Laurenljs G. Crescenzo O ,R . Lei M.A. Mjli!!o PhaUD, PharmacQ/ LeU. 7 (199?) 27-3Q
A Baracco G Bergio E. Gnocco M Legorali S. Sedocco, S CaUnella O EravrellQ, Pt Traldl Raold Commuo M8ss SDectrom 9
119951 427-436
06