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An alys is o f Catech ins and Proanthocyanid ins in G rape Seed s byH P L C w ith P h o t o d i o d e A r r ay D e t e c t io nE. Revil la 1. / M. Bour zeix 2 / E. Alon so 11Universidad Autd noma de Madrid, Departamento de Qu/mica Agr/cola, Geologfa y Geoqufmica, 28049 Madrid, Spain2Institut National de la Recherche Agronomique, Station d'Oenolog ie et de Technologic des Produits V6g6taux, 11100Narbonne, France

Key W ordsColumn liquid chromatographyCatechinsGrape seedsProanthocyanidinsUV-visible spectroscopy/diode array detection

SummaryReversed-phase, high-performance liquid chromatogra-phy coupled to photodiode array detection has been used toanalyse catechins and proanthocyanidins extracted fromgrape seeds. Results show that the ethyl acetate fractionobtained by passing extracts of samples adjusted to pH 7.0through precondit ioned C~8 SEP-PAK cartridges conta insseveral catechins and proanthocyanidins. Seven peaks havebeen assigned to standard catechins and proanthocyanidinson the basis of their retention times and UV spectra. Otherpeaks which appear in the chromatogram show spectralbehaviour similar to that of standard catechins and proan-thocyanidins.

IntroductionGrapes are the edible fruits which probably contain thelargest amounts of catechins (flavan-3-ols) and their oli-gomers, known as proanthocyanidins. The study of thesecompounds in grapes and wines has become a topic ofinterest as a consequence of their positive role in humannutri tion as captors of free radicals [1-2] and in relation tocertain vascular diseases [3]. Catechins and p roanthocya-nidins are more abundant in lignified tissues than in otherplant tissues; thus, grape seeds and cluster stems are richerin them than grape skins and pulps [4-5]. The amount ofcatechins and proanthocyanidins in the different parts ofthe grape cluster greatly affects the content of those sub-stances in wines, which is in turn dramatically influenced by

winemakingprocedures [4]. Nevertheless, a variable amountof catechins and proanthocyanidins remains in grape seedsafter winemaking, depending on the technological processesused; hence, this winemaking by-product may be used as asource of catechins and proanthocyanid ins for pharmaco-logical and cosmetic purposes.Traditionally, grape and wine phenolics have been separat-ed by conventional techniques, such as paper, thin-layerand column chromatography, which are time-consumingand do to permit quantitative analysis. In recent years,reversed-phase, high-performance liquid chromatographyhas proved to be a useful technique for the analysis ofphenolic compounds, and several methods have been de-veloped to analyse catechins and proanthocyanidins ingrapes, in wines, and in other beverages [5-9]. In order toavoid interference by other phenolic compounds in HPLCand also to reduce the time of analysis, several attemptshave been made to perform a simple and rapid fractiona-tion of grape and wine phenolics before HPL C analysis [10-12].The UV-visible spectra of catechins and proanthocyanidinsgive an absorption maximum between 270 and 280 nm,which is remarkably differen t to those of most plan t phe-nolics which present two or more absorption maxima be-tween 250 and 400 nm [13-14]. Some phenols and phenolicacids with very simple substitution patterns are the only on eto present a unique absorption maximum between 270 and280 nm [15]. Thus, if these substances are removed f rom thesample before HPLC analysis, the UV-visible spectra ofpeaks in the chromatogram may be used for their identifi-cation. For these reasons, photodiode array detectioncoupled with HPLC may permit rapid and simultaneousseparation, identification and quantitation o f catechins andproanthocyanidins after the fractionation of grape andseed phenolics.In this paper, a liquid chromatograph equipped with aphotodiode array detector was used to analyse catechinsand proanthocyanidins extracted from grape seeds afterpurification of the extracts by reversed-phase silica mini-columns, and to achieve the spectral purity of differentpeaks in the chromatogram.

Chromalographia Vol. 31, No. 9110, May 1991 Originals 4650009-5893/91/5 0465-04 $ 03.00/0 9 1991 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

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E x p e r i m e n t a lPreparation of Sam plesC h a r d o n n a y g r a p e s w e r e c o l l e c t e d a t P e c h R o u g e E x p e r i -m e n t a l V i n e y a r d ( I N R A , F r a n c e ) d u r i n g t h e 1 9 89 h ar v e s t .O n c e i n t h e l a b o ra t o r y , g r a p e s e e d s w e r e s e p a r a t e d f r o mp u lp , an d d r i ed u n d er CO 2 t o e l imin a t e su r f ace wa t e r . 1 0 .0 0 gs e e d s w e r e p l a c e d i n a f la s k a n d 4 0 m L m e t h a n o l w a sa d d e d . T h e n , s a m p l e s w e r e p u l p e d u n d e r C O 2 w i t h a n U 1-t r a - T u r r a x T 2 5 m i x e r , a n d f l a s k s k e p t a t - 2 4 ~ f o r o n en i g h t. A f t e r w a r d s , t h e s a m p l e s w e r e c e n t r i f u g e d a t 30 0 0 gf o r 1 0 r a in i n a J o u a n G R 2 0 0 0 S c e n t r i f u g e a n d t h e s u p e r -n a t a n t l i q u i d s w e r e s t o r e d . T h e r e s i d u e s w e r e t h e n , e x t r a c t -e d s e q u e n t ia l l y w i t h 80 % m e t h a n o l a t r o o m t e m p e r a t u r ef o r 4 h o u r s , w i t h 50 % m e t h a n o l a t r o o m t e m p e r a t u r e f o r 4h o u r s , w i t h di s t il l e d w a t e r a t - 2 4 ~ f o r 1 5 h o u r s a n d w i t h7 5 % a c e t o n e a t r o o m t e m p e r a t u r e f o r 1 h o u r. A f t e r w a r d s ,a l l t h e e x t r a c t s w e r e m i x e d a n d t h e v o l u m e r a i s e d t o 2 5 0m L . L a s t ly , t h e m e t h a n o l w a s r e m o v e d f r o m 2 5 m L a l i q u o tsb y v a c u u m a t 3 0 ~ a n d t h e ir v o l u m e r e s t o r e d to 2 5 m Lwi th d i s t i l l ed wa t e r .Fractionation of Phenolic CompoundsT h e f r a c t i o n a ti o n o f p h e n o li c c o m p o u n d s w a s c a r r i e d o u to n t w o W a t e r s C 18 S E P - P A K c a r t ri d g e s c o n n e c t e d b y r u b -b e r t u b e ( p r e c o n d i t i o n e d b y s e q u e n t i a l l y p a s si n g 1 0 m Lm e t h a n o l a n d 2 . 5 m L d i s t il l e d w a t e r a d j u s t e d t o p H 7 . 0 w i t hN a O H d r o p w i s e ) , b y i n je c t in g 0 .5 m L o f t h e a l c o h o l - fr e es a m p l e o f t h e g r a p e s e e d e x t r a c t a d j u s t e d t o p H 7 . 0 t oa b s o r b n e u t r a l p h e n o l i c s . T h e n , 1 0 m L d i s t il l e d w a t e r a d -j u s t e d t o p H 7 . 0 w a s p a s s e d t h r o u g h t h e c a r t r i d g e s t o e l u t ea c i d i c p h e n o l i c c o m p o u n d s . T h e c a r t r i d g e s w e r e d r i e du n d e r N 2 , a n d 1 0 m L e t h y l a c e t a t e p a s s e d t h r o u g h t h e m t oe l u t e c a t e c h i n s a n d p r o a n t h o c y a n i d i n s . A f t e r w a r d s , t h ec a r t r i d g e s w e r e w a s h e d b y s e q u e n t i a l l y p a s s i n g 2 .5 m Ld i s ti l le d w a t e r a d j u s t e d t o p H 2 . 0, 2 .5 m L 1 6 % a c e t o n i t r i l ea d j u s te d t o p H 2 .0 , a n d 1 0 m L m e t h a n o l t h r o u g h t h e m t oe l u t e o t h e r p h e n o l i c s , m a i n l y a n t h o c y a n i n s a n d r e d a n db r o w n p o l y m e r s . T h e e t h y l a c e t a t e f r a c t io n w a s d r i e d u n d e rv a c u u m a t 3 0 ~ a n d t h e r e s i d u e d i s s o l v e d i n 0 .5 m L 5 0 %m e t h a n o l a n d s t o r e d 0 ~H P L C A n a l y si sA H e w l e t t - P a c k a r d l i q u i d c h r o m a t o g r a p h e q u i p p e d w i t ha n H I ) 1 05 0 s e r ie s q u a t e r n a r y p u m p , a R h e o d y n e 7 1 2 5i n j e c t i o n v a l v e f u r n i s h e d w i t h a 2 0 g L s a m p l e l o o p , a n H P1 0 4 0 A d i o d e a r r a y d e t e c t o r a n d a n H P 9 0 0 0 s e r i e s 3 0 0c o m p u t e r w a s u s e d . T h e s e p a r a t i o n o f c a t e c hi n s a n d p r o a n -t h o c y a n i d i n s w a s c a r r i e d o u t w i t h a n A l t e c h c o l u m n( 1 00 x 2 .1 r a m ) p a c k e d w i t h 3 0 - 3 8 g m C O : P E L L O D S

Table I Solvent gradient used in HP LC analysis.Time (minutes) % A % B

0 10 9047 82 1855 100 065 100 0

( W h a t m a n L t d ., E n g l a n d ) a n d a p r e - p a c k e d a n a l y t ic a lc o l u m n ( 2 5 0 x 4 .6 m m ) o f N u c l e o s i l C t 8 , 5 # m ( S . E C . C . ,Neu i l l y , F ran ce) , u s in g a l i n ea r g r a d i en t o f 1 0 % ace t i c ac id( s o l v e n t A ) i n w a t e r ( s o l v e n t B ) , a s s h o w n i n T a b l e I , a t af l o w r a t e o f 0 .8 m L r a i n - 1. C o l u m n s w e r e p l a c e d i n a w a t e rb a t h a t 3 2 ~T L C A n a l y s isI n d u b i o u s c a s e s , t h e e f f l u e n t s c o r r e s p o n d i n g t o s e v e r a lp e a k s i n th e c h r o m a t o g r a m s w e r e c o l l e ct e d , to f o l l ow t h e i rb e h a v i o u r i n t w o - d i m e n s i o n a l c e l lu l o s e M N - 3 0 0 T L C , u s-i n g 2 0 x 2 0 c m p l a t e s , w h i c h w e r e d e v e l o p e d w i t h t e r t -b u t a n o l / a c e t i c a c i d / w a t e r ( 3 :1 :1 v / v ) a n d w a t e r / a c e t i c ( 8 5: 15v / v) . T h e c o m p o n e n t s w e r e d e t e c t e d b y s p r a y i n g th e p l a t e sw i t h 1 % p - v a n i l l i n i n c o n c e n t r a t e d h y d r o c h l o r i c a c id , gi v-i n g p i n k - c o l o u r e d s p o t s .S t a n d a r d s( + ) - c a t e c h i n a n d ( - ) - e p i c a t e c h i n w e r e f r o m F l u k a A S( S w i t z e r l a n d ) a n d A l d r i c h C h e m i c a l C o . ( U S A ) , r e s p e c -t iv e ly . P r o c y a n i d i n s B 1 , B 2 , B 3 , B 4 a n d C 1 w e r e s u p p l i e d b yD r . M . M o u n t o u n e t , I n s t it u t d e P r o d u i t s d e l a V i g n e ,M o n t p e l l i e r , F r a n c e .

R e s u l t s a n d D i s c u s s io nT h e c h r o m a t o g r a p h i c a n a ly s i s o f s o l u t i o n s c o n t a i n i n g ( + ) -c a t e c h i n , ( - ) - e p i c a t e c h i n , a n d p r o c y a n i d i n s B 1 , B 2 , B 3 , B 4

L C 2 8 0 n m o r C H FI P, ,D O N NF IY S E E D S . 2 5 . 0 0 - 3 7 . O 0 m ~ n .1614

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413 45 5{3 55 60T t m a / m i r a . )Figure 1Chrom atogram of Chardonnay grape seeds extracts recorded at 280r im .

466 Chro ma tograp hia Vol . 31 , No. 9 /10 , Ma y 1991 Orig ina ls

8/8/2019 Analysisi of Catechin in Grape Seed Extract

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a n d C 1 a l lo w s a c o n v e n i e n t s e p a r a t i o n o f a l l t h e s e s u b s t a n c e s ,w h o s e r e t e n t i o n t i m e s a r e l i s t e d i n T a b l e I I . A l l t h e p e a k s i nt h e c h r o m a t o g r a m s s h o w s i m i la r U V s p e c t r a a t t h e u p s lo p e ,a t t h e a p e x a n d a t t h e d o w n s l o p e o f t h e p e a k i n th e r a n g e2 50 to 4 0 0 nm . A b s o r p t i o n m a x i m a w e r e c l o s e t o 27 7 n m i ne a c h c a s e , a n d s p e c t ra l p e a k p u r i t y w a s g r e a t e r t h a n 9 5 % .T o s t a n d a r d i s e t h is m e t h o d o l o g y f o r q u a n t i t a t i v e e s t i m a t i o no f c a t e c h i n s a n d p r o a n t h o c y a n i d i n s i n p l a n t e x t r a c t s , f o o d s ,a n d b e v e r a g e s ; so l u t io n s o f t h e a b o v e - m e n t i o n e d c o m -p o u n d s a t i n c re a s i n g c o n c e n t r a t i o n s w e r e i n j e c t e d i n t o t h ec h r o m a t o g r a p h . T h e c a l i b r a t i o n c u r v e s , g i v i n g t h e p e a ka r e a s a g a i n s t t h e c o n c e n t r a t i o n o f t h e s o l u t i o n s i n j e c t e d ,a r e s t r a i g h t l i n e s f o r a ll c o m p o u n d s .C h r o m a t o g r a m s o f th e e x t r a c ts o f C h a r d o n n a y g r a p e s e e d ss h o w t h e p r e s e n c e i n t h a t m a t e r i a l o f a n u m b e r o f c o m -p o u n d s w h o s e c h r o m a t o g r a p h i c a n d s p e c t r a l c h a r a c te r i st i csa r e c l o se t o t h o s e o f s t a n d a r d c a t e c h in s a n d p r o a n t h o c y a -n i d i n s . F i g u r e 3 s h o w s t h e c h r o m a t o g r a m [ a t 2 8 0 n m ] b e -t w e e n 2 5 a n d 3 7 m i n a n d b e t w e e n 3 7 a n d 6 3 r a i n a n d a t l e a s t2 2 d i f f e r e n t p e a k s c a n b e s e e n . T h e f o u r m a j o r p e a k s ( 8, 9,1 5 a n d 2 1 ) w e r e a s s i g n e d t o p r o c y a n i d i n B 1 , ( + ) - c a t e c h i n ,p r o c y a n i d i n B 2 a n d ( - ) - e p i c a t e c h i n , r e s p e c t iv e l y , o n t h eb a s is o f t h e i r r e t e n t i o n t i m e s a n d U V - v i s i b le s p e c t r a . T h eU V - v i s i b l e s p e c t r a b e t w e e n 2 5 0 a n d 4 00 n m o f ( + ) - c a t e c h i n ,p r o c y a n i d i n B 2 , p e a k 9 a n d p e a k 1 5 a r e i n F i g u r e 4 a n d , a ss e e n , t h e i r s p e c t r a a r e c l o s e t o t h o s e o f s t a n d a r d m a t e r i a l s .R e t e n t i o n t i m e s a n d s p e c t r a o f a n o t h e r t h r e e p e a k s ( 4, 1 0a n d 2 2 ) a r e c l o s e t o t h o s e o f p r o c y a n i d i n B 3 , p r o c y a n i d i nB 4 a n d p r o c a n i d i n C 1 , a n d m a y b e a s s i g n e d t o t h e s e s u b -s t a nc e s . T h e s p e c t r a l p e a k p u r i t y o f t h e s e s e v e n p e a k s w a sg r e a t e r t h a n 9 5 % . F u r t h e r a n a l y s is i n v o l v in g T L C c o -c h r o m a t o g r a p h y o f t h e e f f l u e n ts c o n t a in i n g t h e s e p e a k sw i t h s t a n d a r d p r o a n t o c y a n i d i n s s h o w s t h e i r i d e n t i t y w i t ht h e m .S o m e o t h e r p e a k s s h o w s p e c t r a l c h a r a c t e r i s t i c s c l o s e t ot h o s e o f c a t e c h i n s a n d p r o a n t o c y a n i d i n s : t h e i r U V X max i sc l o s e to 2 8 0 n m , a s s h o w n i n F i g u r e 5 f o r p e a k s 2 a n d 3 , t h e i ra b s o r p t i o n s p e c t r a d o n o t p r e s e n t a n y o t h e r ) ~rnax a t h i g h e rw a v e l e n g t h s a n d t h e s p e c t ra l p e a k p u r i t y w a s g r e a t e r t h a n9 0 % . T L C a n a ly s is c a r r ie d o u t w i t h t he H P L C e f f l u e n t o fp e a k 1 d e m o n s t r a t e s t h a t i t c o n t a in s a c o m p o u n d w h o s eb e h a v i o u r i n t h e T L C s y s t e m u s e d i s s i m i l a r to t h a t o f t h ec a t e c h i n s a n d t h e p r o a n t h o c y a n i d i n s a v a i l a b l e i n o u r l a b -o r a t o r i e s , g i v i n g a p i n k - c o l o u r e d s p o t w h e n t h e p l a t e w a ss p r a y e d w i t h 1 % p - v a n i l l i n i n c . H C I . T h u s , t h e m a j o rs u b s t a n c e c o n t a i n e d i n p e a k 1 p r o b a b l y b e l o n g s t o t h i s

T a b l e ! 1 R e t e n t i o n t i m e s o f s o m e c a t e c h i n s an d p r o a n t h o -c y a n i d i n s .

Substance Relative retention times(minutes)procyanidin B3 31.6procy anidin B1 35.0(+)-catechin 38.2procyanidin B4 38.3procyanidin B2 44.6(-)-epicatechin 56.2procya nidin C1 60.9

1 8 0 1 / % ( + ) - C R T E C H I N

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9 , 3 0 3 - = ; 0 4 8 81 . 4 a v e . 1 e . n _ q t . h ( , T r n )

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t2 , e lo t \ . . . . . .3 o L 3 3 5 ~

1 , 4 a v e 1 e i ' t g t - t 7 ( r , r t ' ,), , ' 7 ' " , I4 8 0

4811 Pe~ k 15

3 1 ~ O 3 5 ~ 4 0 ~N a , , e l e i -, g t .l - , ~ r,m)F i g u r e 2UV-visible spectra of (+)-catechin, procyanidin B2, peak 9 and peak 15.

P e a k 2,

t 0 . f - - . .

3 0 0 3 5 8 4 0 06 1 a ,~ I e n g t h ( n m )

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15-1

0 . . . . , . . . . ,3 0 01 4 a ve I e n g t h ( r i m )F i g u r e 3UV-visible spectra o f peaks 2 and 3.

, , , , - , - - i~ 5 0 4 0 0

Chro ma tograph ia Vol . 31 , No. 9 /10 , May 1991 Orig inals 467

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f a m i l y o f f l a v o n o i d s b u t i s p r o b a b l y d i f f e r e n t t o t h o s er e c e n t l y i d e n t i f i e d b y L e e a n d J a w o r s k y [ 16 ] b e c a u s e i tsr e t e n t i o n t i m e i s l e ss t h a n f o r s t a n d a r d c a t e c h i n s a n d p r o a n -t o c y a n i d i n s a v a i l a b l e a n d t h e s u b s t a n c e s i d e n t i f i e d b y t h e s ea u t h o r s h a v e r e t e n t i o n t im e s h i g h e r t h a n p r o c y a n i d i n B 1 .H o w e v e r , f u r th e r e x p e r i m e n t s a r e n e e d e d t o i s o l a te t h e mi n s u f f i c ie n t q u a n t i t i e s t o d e t e r m i n e t h e i r s t r u c t u r a l c h a r a c -ter i s t ics .

Tab le 111 Catechin and proanthocyanidin content of Chardonnaygrape seeds (mg kg-1). Results are m eans of three sam ples.Substance Mean value(nag kg 1)

(+)-catechin 3012(-)-epicatechin 4175procyanidin B1 708procyanidin B2 767procyanidin B3 679procanidfn B4 592procya nidin C1 979

T h e q u a n t i t a t i v e a n a l y s i s o f c a t e c h i n s a n d p r o a n t h o c y a n i -d i ns c o n t a i n e d i n th e e x t r a c ts o f C h a r d o n n a y s e e d s s h o w st h a t t h i s m a t e r i a l c o n t a i n s m a i n l y c a t e c h i n s , a s s h o w n i nT a b l e I l l . P r o a n t h o c y a n i d i n s a r e p r e s e n t t o a l e s se r e x t e n ta n d p r o c y a n i d i n C 1 s e e m s t o b e t h e m a j o r o n e . T h e t o t a lc o n t e n t o f c a t e c h in s a n d p r o a n t h o c y a n i d i n s o f C h a r d o n n a ys e e d s s h o u l d b e o v e r 1 0 g k g 1 . T h e s e r e s u l t s a r e v e r y c l o s et o t h o s e r e p o r t e d b y K o v a c e t a l. [1 7] f o r s e v e r a l r e d a n dw h i t e g r a p e c u l t i v a r s . T h e m a j o r c o m p o n e n t s s h o w l o wc o e f f i c i e n t s o f v a r ia t i o n , w h i c h a r e h i g h e r f o r t h e m i n o rc o m p o n e n t s , a n d e s p e c i a ll y f o r th o s e w h o s e p e a k s a r e n o tc o m p l e t e l y r e s o l v e d ( p r o c y a n i d i n s B 3 , B 4 a n d C 1 ) .

ConclusionsT h e r e s u lt s p r e s e n t e d d e m o n s t r a t e t h e s u i ta b i li t y o f U V -v i s i b l e , p h o t o d i o d e a r r a y d e t e c t i o n f o r q u a l i t a t i v e a n dq u a n t i t a ti v e H P L C a n a ly s is o f c a te c h i n s a n d p r o a n t h o c y -a n i d i n s o f g r a p e s e e d s . T h e r e c o r d i n g o f U V - v i s i b le s p e c t r ad u r i n g c h r o m a t o g r a p h i c a n a l y s i s s h o w s t h e s p e c t r a l c h a r -a c t e r is t i c s o f t h e d i f f e r e n t p e a k s i n t h e c h r o m a t o g r a m a n ds h o u l d p e r m i t a m o r e c o m p l e t e i d e n t i f ic a t i o n o f th e d i f f e r e n tc a t e c h in s a n d p r o a n t h o c y a n i d i n s p r e s e n t i n th e s a m p l e a n dt h e d e t e c t i o n o f o t h e r p h e n o l i c c o m p o u n d s w i t h d i f f e r e n tU V - v i s i b l e s p e c t r a , i f p r e s e n t .T h e p r e s e n c e i n C h a r d o n n a y s e e d s o f s e v e r a l c a te c h i n s a n dp r o a n t h o c y a n i d i n s d e s c r i b e d p r e v i o u s l y i n g r a p e s e e d s [ 5]

i s c o n f i r m e d b y c o m p a r i s o n o f th e i r U V - v i s i b l e s p e c t r a w i tht h o s e o f s t a n d a r d s u b s t a n c e s . I n a d d i t i o n , o t h e r c o m p o -n e n t s p r e s e n t i n t h e s e s a m p l e s s h o w s p e c t r a q u i t e s i m i l a r t ot h o s e o f s t a n d a r d c a t e c h i n s a n d p r o a n t h o c y a n i d i n s . T h u s ,t h e y m a y b e l o n g t o t h e s e g r o u p o f f l a v o n o id s b u t f u r t h e re x p e r i m e n t s a r e n e e d e d t o e l u c i d a t e t h e ir s t r u c tu r a l c h a r-ac t e r i s t i c s .

AcknowledgementsT h e a u t h o r s a c k n o w l e d g e f i n a n ci a l s u p p o r t f r o m I n s ti t utN a t i o n a l d e l a R e c h e r c h e A g r o n o m i q u e ( F r a n c e ) , a n df r o m M i n i s t e r io d e E d u c a c f o n y C i e n c i a ( S p a i n) a n d M i n -i s t 6 r e d e l a R e c h e r c h e e t l a T e c h n o l o g i e ( F r a n c e ) i n t h ef o r m o f a M E C / M R T p o s t d o c t o r a l f e ll o w s h ip f o r o n e o f us( E . A . ) .

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Received: N ov. 20, 1990Revised manuscriptreceive d: Jan. 17, 1991Accepted: M arch 3, 1991D

468 Chr om atogr aphia Vol . 31 , No. 9 /10 , Ma y 1991 Orig inals