Quantitative Analysis of Polymeric Procyanidins (Tannins) from

Grape (Vi ti s vi ni fera) Seeds by Reverse Phase High-Performance

Liquid Chromatography
Zhongkui Peng,*
,,
Yoji Hayasaka,
,
Patri ck G. I l and,
,
Mark Sefton,
,
Peter Hj,
,,
and
El i zabeth J. Waters
,
Cooperati ve Research Centre for Vi ti cul ture, P.O. Box 154, Gl en Osmond, South Austral i a 5064, Austral i a;
The Austral i an Wi ne Research I nsti tute, P.O. Box 197, Gl en Osmond, South Austral i a 5064, Austral i a; and
Department of Horti cul ture, Vi ti cul ture and Oenol ogy, Facul ty of Agri cul tural and Natural Resource
Sci ences, Uni versi ty of Adel ai de, Gl en Osmond, South Austral i a 5064, Austral i a
A reverse phase C
18
HPLC method wi th potenti al for hi gh automated throughput has been devel oped
for the quanti tati ve anal ysi s of pol ymeri c procyani di ns (tanni ns) i n grape seed extracts. Chroma-
tography gave ri se to 13 di sti nct UV-absorbi ng peaks wi th good basel i ne separati on. The
UV-absorbi ng peak el uti ng l ast i s di sti nct and therefore easi l y quanti fi ed. Bi ochemi cal anal yses
i ncl udi ng ul trafi l trati on, protei n preci pi tati on, and Sephadex LH20 chromatography combi ned wi th
el ectrospray mass spectrometri c anal yses establ i sh that thi s peak predomi nantl y contai ns pol ymeri c
procyani di ns. The pol ymers, whi ch appear to be gal l oyl ated to vari ous degrees and seem to fragment
i n a characteri sti c manner duri ng el ectrospray mass spectrometry, are wel l separated from catechi ns
and procyani di n ol i gomers of up to 4 uni ts. The recovery of pol ymeri c grape seed tanni ns wi th thi s
HPLC method was 86%, whi ch i s si mi l ar to the 89% recovery achi eved wi th commerci al quebracho
tanni ns. The concentrati on of tanni ns i n seeds from ri pe Vitis vinifera cv. Shi raz grapes ranged
from 1360 to 2830 mg/kg of berri es.
Keywords: Proanthocyanidin; liquid chromatography electrospray mass spectrometry; Sephadex
LH20 chromatography; ultrafiltration; protein precipitation; PVPP; Shiraz; Pinot Noir
I NTRODUCTI ON
Procyani di ns consti tute the major cl ass of phenol i c
compounds i n grape seeds and chi efl y compri se fl avan-
3-ol pol ymers wi th an average degree of pol ymeri zati on
(DP) rangi ng from 2 to >15 and an average mol ecul ar
mass rangi ng from 578 to >5000 Da (see structure;
1-3). Grape seed pol ymeri c procyani di ns (tanni ns) are
extracted duri ng the l atter stages of wi ne-maki ng and
are bel i eved to contri bute to col or stabi l i ty and organo-
l epti c properti es of wi ne (4-6). I t i s not cl ear, however,
what the rel ati ve i mpact of monomers, ol i gomers (here
defi ned as DP e 4), or pol ymers (here defi ned as DP g
5) i s i n thi s regard and, i ndeed, how speci fi c vi ti cul tural
and oenol ogi cal ci rcumstances contri bute to the modul a-
ti on of the rel ati ve abundance of these vari ous speci es.
Knowl edge i n thi s area woul d be greatl y assi sted by a
faci l e method for the rapi d measurement of monomers,
ol i gomers, and pol ymers of the seed and thi s coul d i n
turn l ead to better i nformed deci si on-maki ng about
vi ti cul tural and wi ne-maki ng practi ces.
Reverse phase hi gh-performance l i qui d chromatog-
raphy i s an effecti ve and accurate techni que for the
anal ysi s of catechi ns and ol i gomeri c procyani di ns (e.g.,
refs 5-9). However, reports on the use of thi s techni que
i n the anal ysi s of pol ymeri c procyani di ns are few and
have not been compl emented by the more recent tech-
ni que of el ectrospray i oni zati on technol ogy so el egantl y
pursued by Cheyni er and col l eagues for the character-
i zati on of a vari ety of proanthocyani di ns (e.g., 10-13).
I n previ ous studi es of grape seed composi ti on usi ng
HPLC, a very broad UV-absorbi ng peak el uti ng l ate i n
the chromatogram was thought to represent a mi xture
of pol ymeri c pol yphenol i c compounds (4, 14-17). The
broad nature of the suspected pol ymeri c peak i n those
studi es makes i t hard to quanti fy. I n thi s study we
descri be a method whereby a di sti nct and easy to
quanti fy peak compri si ng mostl y, i f not excl usi vel y,
pol ymers i s cl earl y resol ved from previ ousl y i nterferi ng
* Correspondi ng author (tel ephone +61 8 8303 6634; fax
+61 8 8303 6601; e-mai l zpeng@awri .adel ai de.edu.au).

Cooperati ve Research Centre for Vi ti cul ture.

The Austral i an Wi ne Research I nsti tute.

Uni versi ty of Adel ai de.

26 J. Agric. Food Chem. 2001, 49, 2631
10.1021/jf000670o CCC: $20.00 2001 American Chemical Society
Published on Web 12/15/2000
monomers and ol i gomers. The techni que has been
empl oyed to determi ne l evel s of procyani di n pol ymers
i n seeds from grapes grown i n commerci al Austral i an
vi neyards.
MATERI ALS AND METHODS
Chemical Standards. The chemi cal s used were of the
hi ghest puri ty commerci al l y avai l abl e. Gal l i c aci d and catechi n
standards were obtai ned from Fl uka Chemi e AG (Buchs,
Swi tzerl and) and epi catechi n, epi catechi n gal l ate, and pol y-
vi nyl pol ypyrrol i done (PVPP) from Si gma-Al dri ch (Sydney,
Austral i a). The commerci al quebracho tanni n (Quebracho
UNI TAN ATO) was a gi ft from Redox Chemi cal s Pty. Ltd.,
South Austral i a.
PreparativeSephadex LH20Chromatography. Sepha-
dex LH20 gel (Pharmaci a Bi otech AB, Uppsal a, Sweden),
preswol l en i n 60% (v/v) methanol and 0.2% (v/v) formi c aci d,
was sl urry-packed i nto a gl ass col umn (3.5 16 cm); pri or to
sampl e l oadi ng, the col umn was equi l i brated wi th Mi l l i -Q
water. The ethanol extracts of grape seeds were l oaded onto
the col umn, and the col umn was washed sequenti al l y wi th 2.3
L of 60% (v/v) methanol pl us 0.2% (v/v) formi c aci d and 2.3 L
of Mi l l i -Q water at a fl ow rate of 0.3-0.5 mL/mi n. Pol ymeri c
procyani di ns were el uted wi th 2.3 L of 60% (v/v) acetone pl us
0.2% (v/v) formi c aci d. Acetone was evaporated wi th a rotary
vacuum evaporator at 35 C. The resi dual sampl e was l yoph-
i l i zed and further dri ed i n a desi ccator at room temperature
for >24 h. The puri ty of seed tanni n was exami ned by HPLC
anal ysi s (see bel ow).
Source of Grape Samples. For HPLC method devel op-
ment, heal thy Vitisvinifera cv. Shi raz, Pi not Noi r, and Muscat
of Al exandri a grapes from the 1997 vi ntage wi th total sol ubl e
sol i ds of between 22.7 and 24.7 Bri x were harvested from
commerci al vi neyards i n Austral i a and stored at -20 C pri or
to extracti on.
Extraction. Seeds were manual l y separated from 100
berri es and homogeni zed i n a hi gh-speed homogeni zer (Ul tra-
turrax T25, Janke & Kunkel GmbH & Co.) wi th 20 mL of 70%
(v/v) aqueous ethanol and extracted wi th occasi onal manual
shaki ng at room temperature for 1 h. The sl urry was centri -
fuged (12000g, 5 C, 40 mi n), and the preci pi tate was re-
extracted twi ce wi th the same sol vent for 30 mi n (2 20 mL).
Supernatants were pool ed, and the vol ume was adjusted such
that 100 mL corresponded to the extract from 6 g of seeds.
HPLC Analysis. The HPLC apparatus used were standard
Waters i nstruments wi th di ode array detectors. Sampl es (20
L), di l uted wi th Mi l l i -Q water (80 L), were l oaded onto a
4.6 250 mm C
18 col umn (Exsi l 100 5 ODS, Acti von, Sydney,
Austral i a) wi th a C18 guard cartri dge wi th the same packi ng
materi al equi l i brated i n sol vent A [0.2%(v/v) phosphori c aci d].
Phenol i c compounds were el uted by a gradi ent of sol vent B
[82% (v/v) acetoni tri l e, 0.04% (v/v) phosphori c aci d] from 0 to
15% sol vent B i n the fi rst 15 mi n, from 15 to 16% from 15 to
40 mi n, from 16 to 17% from 40 to 45 mi n, from 17 to 43%
from 45 to 48 mi n, from 43 to 52% from 48 to 49 mi n, hel d
i socrati c at 52% from 49 to 56 mi n, reduced from 52 to 43%
from 56 to 57 mi n, reduced from 43 to 17% from 57 to 58 mi n,
and reduced from 17 to 0% from 58 to 60 mi n. Peaks were
detected at 280 nm and i denti fi ed by compari son to retenti on
ti mes of standards and as descri bed under Resul ts and
Di scussi on. The concentrati on of the pol ymer peak was quanti -
fi ed by compari son to the peak area of a puri fi ed grape seed
pol ymeri c procyani di n fracti on. The fracti on used as a standard
was the acetone el uate obtai ned by preparati ve LH20 chro-
matography (see above). One hundred mi l l i grams of the
pol ymeri c procyani di n standard was equi val ent to 108 ( 2 mg
of catechi n or 97 ( 8 mg of gal l i c aci dswhen assessed by the
Fol i n-Ci ocateau method (18).
Recovery during HPLC Analysis A seed pol ymeri c
procyani di n standard prepared by preparati ve LH20 chroma-
tography (see above, 100 mg/L) was i njected onto the HPLC
col umn, and sampl es were run ei ther through the col umn
(as descri bed above) or through a bypass (the col umn was
repl aced by a short pi ece of stai nl ess steel tubi ng). The el uti ng
sol vent for seed pol ymeri c procyani di ns run through a bypass
was 42:58 acetoni tri l e/sol vent Asthe equi val ent sol vent under
whi ch the pol ymeri c fracti on el utes when run through the
col umn. The rati o of the peak area obtai ned through the
col umn to that obtai ned through a bypass was used as an
i ndi cati on of the recovery of the pol ymeri c procyani di n. A
preparati on of commerci al quebracho tanni n sol uti on (500 mg/
L) was al so tested.
Ultrafiltration. A regenerated cel l ul ose ul trafi l trati on
membrane (Mi crocon 3, Ami con I nc., Beverl y, MA) of 3000
nomi nal mol ecul ar wei ght cutoff (NMWCO) was used. Aci di fi ed
aqueous sampl es [contai ni ng 0.2% (v/v) formi c aci d] of grape
seed extracts were centri fuged (12000g, 5 mi n) to remove
sol i ds. The supernatant of 0.5 mL was pl aced i n each cel l and
centri fuged (10000g, 45 mi n) unti l the retentate vol ume was
reduced to 0.1 mL. The fi rst fi l trate was di rectl y used for
HPLC anal ysi s. The vol ume of the retentate was adjusted to
0.5 mL by the addi ti on of 0.4 mL of 0.2% (v/v) formi c aci d.
After gentl e mi xi ng, sampl es were further centri fuged as
descri bed above. The ul trafi l trati on was conducted three ti mes,
and after adjustment to i ts ori gi nal vol ume, the fi nal retentate
was anal yzed by HPLC.
PVPP Assays. A 0.9 7 cm gl ass col umn was packed wi th
1.2 g of cl eaned and dry PVPP powder (Si gma-Al dri ch, Sydney,
Austral i a). Approxi matel y 12 mL of the prepared seed extracts
of Shi raz grapes was l oaded and fi l tered under vacuum. Two
mi l l i l i ters of the fi l trate was col l ected after the fi rst 200 L
had been di scarded. Sampl es of the PVPP-treated seed extracts
and the untreated control s were anal yzed by HPLC.
BSAAssays. Approxi matel y 40 mL of 70%ethanol extracts
of Shi raz grape seeds was dri ed i n a rotary evaporator at 45
C, redi ssol ved i n 4 mL of 12.5%(v/v) ethanol , and centri fuged
(10000g, 5 C, 40 mi n). The concentrati on of pol yphenol i c
compounds i n the supernatant was adjusted to 8000 mg/L
gal l i c aci d equi val ents and added to an equal vol ume of 8000
mg/L BSA (Si gma Pty. Ltd.) i n 12.5% (v/v) ethanol , and the
mi xture was shaken thoroughl y. An equal vol ume of the seed
preparati on and 12.5% (v/v) ethanol was mi xed and used as
control s. Both treatments and control s were centri fuged
(12000g, 10 mi n, room temperature) after an i ncubati on at -7
C for 4 h. The supernatants (3 mL) were col l ected, and 10%
(v/v) tri chl oroaceti c aci d was added (0.3 mL) pri or to HPLC
anal ysi s i n tri pl i cate.
LiquidChromatography Electrospray MassSpectrom-
etry (LC-ESI/MS) Analysis. An extract of Shi raz grape seed
phenol i c compounds was concentrated at 45 C usi ng a rotary
vacuum evaporator before anal ysi s on a mass spectrometer
(API -300, PE Sci ex, Thornhi l l , ON, Canada). The extract was
l oaded through a 5 L sampl e l oop onto an HPLC C18 col umn
(Nova-Pak, 60 , 4 m, 2 150 mm, Waters). The separati on
was carri ed out usi ng a fl ow rate of 100 L/mi n and a gradi ent
the same as that descri bed above except that 0.2%(v/v) formi c
aci d repl aced 0.2%(v/v) phosphori c aci d to i mprove the si gnal -
to-noi se rati o. The el uant was spl i t (1:4) postcol umn by a tee,
at a fl ow rate of 20 L/mi n to the mass spectrometer and 80
L/mi n to a UV detector (HP1100, Hewl ett-Packard) moni tor-
i ng at 280 nm.
The mass spectrometer was operated under posi ti ve i on
mode and scanned from m/z 250 to 3000 wi th a mass step si ze
of 0.2 Da and a dwel l ti me of 0.5 ms. The curtai n (ni trogen)
and nebul i zer (ai r) gases were set at 8 and 10 uni ts, respec-
ti vel y. The i on spray vol tage was 5500 V, and the ori fi ce
potenti al was 40 V. Mass spectral data were processed usi ng
Bi o-Mul ti vi ew software 1.2 v 3 (PE Sci ex).
Spectrophotometric Analysis. Grape seed extracts (1
mL) were heated for 40 mi n at 95 C wi th 6 mL of aci d reagent
[70% (v/v) concentrated HCl i n butanol ] i n the presence of 0.2
mL of ferri c reagent [2% (w/v) NH
4Fe(SO4)212H2O (23)]
fol l owed by spectrophotometry at 520 nm. The quanti ty of
procyani di n i n grape seed extracts was determi ned from a
standard curve constructed wi th puri fi ed grape seed pol ymeri c
procyani di n standard (see HPLC Anal ysi s) subjected to the
aci d degradati on procedure.
HPLC Analysis of Polymeric Tannins in Grape Seeds J. Agric. Food Chem., Vol. 49, No. 1, 2001 27
RESULTS AND DI SCUSSI ON
HPLC MobilePhaseDevelopment. I n earl y tri al s
we eval uated publ i shed HPLC methods (4, 15, 17, 19)
for separati on of the phenol i c compounds i n extracts of
seeds from Shi raz or Muscat of Al exandri a grapes. The
separati on of compounds was i n general si mi l ar to that
showed by Guyot et al . (12). Thus, catechi n, epi catechi n,
and epi catechi n gal l ate appeared as smal l peaks on the
shoul der of a broad hump. Thi s broad hump el uted over
a 30 mi n peri od and was thought to represent pol ymeri c
procyani di ns (16). Fol l owi ng extensi ve testi ng and
i nfl uenced by reports i n the l i terature (see, e.g., refs 15
and 18), we devi sed a method wi th a novel sequence of
gradi ents.
The performance of the system i s i l l ustrated i n Fi gure
1a. A wel l -resol ved chromatogram wi th near basel i ne
separati on between most peaks was observed when a
grape seed extract was anal yzed. The sharp defi ni ti on
of peak 13, the procyanidin polymer peak (see bel ow),
was cri ti cal l y dependent on the i ncl usi on of the i socrati c
segment si x (49-56 mi n) at rel ati vel y hi gh acetoni tri l e
concentrati on. Gal l i c aci d, catechi n, epi catechi n, and
epi catechi n gal l ate, represented by peaks 1, 7, 10, and
12, respecti vel y, were i denti fi ed by compari son of thei r
spectral characteri sti cs and retenti on ti mes wi th those
of commerci al standards and by LC-ESI /MS (data not
shown). The i denti ti es of peaks 2-6, 8, and 9 were
assi gned by LC-ESI /MS (see bel ow) and are gi ven i n the
capti on to Fi gure 1. Peak 13 had the same retenti on
ti me as the major peak for commerci al quebracho tanni n
and a grape seed pol ymeri c procyani di n fracti on pre-
pared by Sephadex LH20 chromatography (see bel ow).
Thi s suggested that peak 13 represents grape seed
pol ymeri c procyani di ns.
Confirmation of the Polymeric and Polyphe-
nolic Nature of the Procyani di n Pol ymer Peak
(Peak 13). We subjected grape seed extracts to a
number of standard mani pul ati ons. Fi rst, we compared
the HPLC chromatograms of nonfi ned and PVPP-fi ned
seed extracts. Essenti al l y al l of the peaks, i ncl udi ng the
enti re peak 13, were absent from the chromatogram of
the PVPP-treated sampl e (data not shown), consi stent
wi th al l substances represented by peak 13 bei ng
phenol i c i n nature (20).
Second, grape seed extract was submi tted to fracti on-
ati on usi ng a 3000 NMWCO ul trafi l trati on membrane
(Fi gure 1). Compari son of the chromatograms for the
unfracti onated extract (Fi gure 1a) and retentate (Fi gure
1b) showed that onl y peak 13 was common to the two
chromatograms. Conversel y, the chromatogram ob-
tai ned for the fi l trate was vi rtual l y i denti cal to that of
the whol e extract, except for a >80% reducti on i n the
area associ ated wi th peak 13 (compare parts a and c of
Fi gure 1). The most l i kel y expl anati on i s that a l arge
proporti on, i f not al l , of the materi al associ ated wi th
peak 13 i s pol ymeri c i n nature and cannot pass through
the ul trafi l trati on membrane, whereas gal l i c aci d, the
catechi ns, and procyani di n ol i gomers (peaks 1-12) al l
passed through the fi l ter wi thout a noti ceabl e l oss. I t
i s possi bl e that a smal l amount of pol ymeri c materi al
was formed i n the fi l trate fol l owi ng i ts passage through
the ul trafi l trati on membrane and subsequent storage
pri or to HPLC anal yses or that l ower pol ymers (4 < DP
< 8) were onl y partl y retai ned by the 3000 NMWCO
ul trafi l trati on membrane.
Sephadex LH20 gel fi l trati on chromatography i s
commonl y used for the separati on of both condensed and
hydrol yzabl e tanni ns from si mpl e phenol i c compounds
on a preparati ve scal e (e.g., refs 15 and 21). Catechi ns
and ol i gomeri c procyani di ns are el uted from the Sepha-
dex LH20 col umn by 60% (v/v) methanol , whereas
proanthocyani di n pol ymers are el uted by 60% (v/v)
acetone (15). An HPLC anal ysi s of the methanol el uate
of cv. Muscat of Al exandri a grape seed extracts i s shown
i n Fi gure 2a. Catechi n, epi catechi n, and procyani di n
di mers were the domi nant components i n the methanol
el uants wi th vi rtual l y no peak 13 materi al . Conversel y,
onl y one cl ear peak el uted i n the posi ti on of peak 13
was obtai ned when the acetone el uate was anal yzed
(Fi gure 2b). Later, LC-ESI /MS anal ysi s of the acetone
el uant gave resul ts (data not shown) si mi l ar to those
obtai ned when materi al i n peak 13 from whol e grape
seed extracts was anal yzed.
Figure1. Effect of ul trafi l trati on on the phenol i c composi ti on
of extracts of Shi raz grape seeds. The grape seed extract before
ul trafi l trati on (a), the retentate after ul trafi l trati on through
a 3000 Da NMWCO membrane (b), and the fi l trate after
ul trafi l trati on (c) were anal yzed by RP-HPLC. The peaks were
i denti fi ed pri mari l y by LC-ESI /MS as (1) gal l i c aci d; (2)
procyani di n tri mer (P
3); (3) procyani di n tetramer (P4); (4)
procyani di n di mer (P2); (5) procyani di n di mer (P2); (6) procya-
ni di n tri mer (P3); (7) catechi n; (8) procyani di n di mer (P2); (9)
procyani di n di mer (P2); (10) epi catechi n; (11) monogal l oyl ated
procyani di n di mer (P2G1); (12) epi catechi n gal l ate; and (13)
pol ymeri c procyani di ns. The el uti on of procyani di n tetramers
(P4) as mi nor peaks at 21.5, 25.4, and 29.5 mi n was al so
observed.
28 J. Agric. Food Chem., Vol. 49, No. 1, 2001 Peng et al.
When the grape seed extracts were combi ned wi th
BSA i n 12.5% (v/v) ethanol and hel d at -7 C, the
sol uti on became turbi d. The haze formed coul d be
removed by centri fugati on. HPLC anal yses showed that
the supernatant contai ned l ess of substances el uted as
peak 13 (up to 80%reducti on) compared to the sampl es
wi thout treatment wi th BSA (data not shown). No
si gni fi cant change i n the areas of the other peaks was
observed, and the protei n sol uti on al one di d not gi ve a
si gni fi cant peak under the same condi ti ons. Thi s resul t
i ndi cated that the substances represented by peak 13
had stronger affi ni ty for BSA than di d si mpl e procyani -
di n compounds. Previ ous researchers have suggested
that the affi ni ty of grape seed procyani di ns for protei n
i s determi ned by the number of o-di hydroxyphenyl
groups and i ncreases wi th the DP and gal l oyl ati on (22).
Recovery. The recovery for pol ymeri c procyani di ns
i n peak 13 was 86.4% (n ) 7) for grape seed tanni ns.
The recovery was 89% (n ) 4) when commerci al que-
bracho tanni ns were anal yzed.
QualitativeAnalysesof SeedProcyanidinsSepa-
rated by HPLC. I n-depth descri pti on of i ndi vi dual
mol ecul ar speci es contai ned wi thi n the vari ous peaks
was attempted usi ng LC-ESI /MS. Cheyni er and col -
l eagues have used LC-ESI /MS extensi vel y to i nvesti gate
procyani di ns and have found that detecti on was most
effecti ve when the mass spectrometer was run i n the
negati ve i on mode despi te the aci di c nature of the HPLC
mobi l e phase (10, 11). Wi th our sol vent systems, how-
ever, we found that the total i on counts were hi gher i n
the posi ti ve i on mode and that the predomi nant occur-
rence of the procyani di ns as si ngl y charged speci es,
rather than mul ti pl y charged speci es, as occurs i n
negati ve i on mode, si mpl i fi ed i nterpretati on of the data.
Qualitative Analysis, Peaks 1)12. Assi gnments
of the major peaks, whi ch appeared i n the fi rst stage of
the el uti on (Fi gure 1a), were confi rmed by LC-ESI /MS
anal ysi s (data not shown). The desi gned mobi l e phase
from 0 to 45 mi n el uted nearl y al l of the si mpl e
procyani di ns (monomers and ol i gomers) so far reported
i n grape seeds by HPLC. No si gni fi cant si gnal s of
procyani di n pentamers (m/z 1443) and hi gher DP pro-
cyani di ns were determi ned i n the el uant from 10 to 48
mi n duri ng HPLC anal ysi s. I mportantl y, the l ater peaks
were not surfi ng on a l arge unresol ved hump of mostl y
pol ymeri c materi al .
Qualitative Analyses of the Procyani di n Pol y-
mer Peak (Peak 13). To characteri ze the materi al
associ ated wi th peak 13 further, i t was recovered from
HPLC, concentrated, and subjected to LC-ES/MS anal y-
si s (Fi gure 3). The major si gnal s di stri buted i n the m/z
range of 250-3000 are gi ven (Tabl e 1) and tentati ve
assi gnments made. The major i ons appear to be si ngl y
charged, al though we di d see some evi dence of mul ti pl y
charged speci es such as the si gnal at m/z 1375 (Fi gure
3) for the doubl y charged speci es of a monogal l oyl ated
procyani di n of DP 9 (P
9
G
1
).
Figure 2. Separati on of phenol i c compounds i n Muscat of
Al exandri a grape seed extracts by Sephadex LH20 l ow-
pressure LC. Extracts were l oaded on a Sephadex LH20
col umn as descri bed i n the text and el uted wi th (a) 60% (v/v)
methanol and (b) 60% (v/v) acetone before HPLC anal ysi s.
Figure 3. Spectral data for phenol i c compounds i n the
procyanidin polymer peak of seed extracts determi ned by ESI /
MS. The mass spectrum of the procyanidin polymer peak (peak
13, Fi gure 2a) i s presented i n three m/z ranges: 250-1000,
1000-2000, and 2000-3000. See al so Tabl e 1.
HPLC Analysis of Polymeric Tannins in Grape Seeds J. Agric. Food Chem., Vol. 49, No. 1, 2001 29
Gi ven the bi ochemi cal characteri zati on of peak 13
above and the HPLC behavi or of pure catechi n/epi cat-
echi n, the rel ati vel y l ow m/z val ues of 291, 443, 579,
731, and 867 i n the pol ymer peak requi red some further
attenti on and i nterpretati on. Thei r i sotopi c mass pat-
tern i ndi cates that these l ow mol ecul ar wei ght speci es
are si ngl y charged. Expansi on of the base peak area of
the spectra showed catechi n, the procyani di n di mer, and
procyani di n tri mer al l gave a parent peak (P) of (M +
H)
+
and one (P + 1) or more i sotopi c peaks (P + n) but
no fragments wi th m/z e (P-2) (data not shown). For
i nstance, catechi n wi th a mol ecul ar wei ght of 290 gave
the parent peak at m/z 291 and an i sotopi c peak at m/z
292 but no speci es of m/z e 289. However, when the
m/z 291 regi on associ ated wi th a procyani di n di mer was
expanded and compared to that of the m/z 291 regi on
associ ated wi th catechi n, addi ti onal speci es at m/z 289
and 287 were cl earl y observabl e (not shown). A si mi l ar
pattern was observed when the expanded spectra of
procyani di n di mer-l i ke fragments deri ved from a pro-
cyani di n tri mer were compared to the spectra of a di mer
or si mi l arl y when tri mer-l i ke fragments from procyani -
di n tetramers were compared to a tri mer. I n regard to
the speci es associ ated wi th peak 13, a si mi l ar si tuati on
exi sts and the data i ndi cate that the presence of the
l ower mol ecul ar mass speci es i n the mass spectrum
extracted from peak 13 are consi stent wi th thei r deri va-
ti on from fragmentati on of hi gher mol ecul ar mass
speci es. We are not aware of thi s phenomenon bei ng
reported previ ousl y and can onl y specul ate about the
potenti al mode of formati on.
To l end further support for the contenti on that the
l ower m/z speci es seen i n the spectra extracted from the
pol ymer peak (peak 13) were due to fragmentati on of
l arger pol ymers, the fi l trate from ul trafi l trati on through
the 3000 NMWCO fi l ter was subjected to LC-ESI /MS
anal ysi s. Major peaks at m/z 291 (P
1
), 579 (P
2
), 731
(P
2
G
1
), 867 (P
3
), 1019 (P
3
G
1
), 1155 (P
4
), and 1443 (P
5
)
were extracted from the fi rst phase of the LC anal ysi s
(0-45 mi n). No si gni fi cant si gnal s wi th these m/z val ues
were observed from peak 13 i n the fi l trate [except that
at m/z 1443 (P
5
)], presumabl y because the mol ecul es
previ ousl y gi vi ng ri se to these si gnal s from peak 13 were
retai ned by the ul trafi l trati on membrane and hence of
consi derabl y hi gher mol ecul ar mass than thei r mass
spectra i ndi cated. Thi s resul t supports the asserti on
above that m/z si gnal s <1400 seen wi th peak 13
represented i on fragments from procyani di ns wi th DP
g 4 rather than mol ecul ar i ons.
Al l of the avai l abl e evi dence suggests that peak 13
contai ns l i ttl e or no ol i gomeri c procyani di ns wi th DP e
4 and that the devi sed HPLC method consti tutes a
rel i abl e assay for the pol ymeri c seed procyani di ns.
HPLC Assay of Grape Seeds Sampled from
Commercial Vineyards. The concentrati ons of pol y-
meri c procyani di ns i n grape seeds ranged from 33200
to 50700 mg/kg of seeds or from 1680 to 3190 mg/kg of
grapes. The resul ts obtai ned by HPLC anal ysi s were
l i nearl y correl ated (r
2
)0.96) to the data obtai ned usi ng
the HCl -butanol assay (23), al though the l atter con-
si stentl y gave hi gher val ues, presumabl y because i t
i ncl udes the measurement of procyani di n ol i gomers as
wel l as pol ymers.
I t i s cl ear there i s a l arge degree of vari abi l i ty i n the
content of seed procyani di n pol ymers i n grapes of the
same vari ety at practi cal l y i denti cal sugar maturi ty.
Gi ven that at l east hal f of the tanni ns i n a red wi ne
may be deri ved from seeds (24), pri or knowl edge of the
potenti al pol ymer content of a parti cul ar grape l ot
cl earl y wi l l enabl e wi ne-makers to exert greater choi ce
i n an i nformed manner when al l ocati ng parti cul ar grape
l ots for speci fi c purposes duri ng vi ntage.
Varietal Difference. Seeds from Shi raz grapes from
ei ght di fferent si tes and Pi not Noi r grapes from three
di fferent si tes were anal yzed usi ng the HPLC method.
The rati os of pol ymers to monomers (catechi n pl us
epi catechi n) were 8.9 ( 1.4 (n ) 9) for Shi raz and 5.2 (
1.9 (n ) 13) for Pi not Noi r, whi ch are consi stent wi th
the fi ndi ngs of Thorngate and Si ngl eton (18), who
detected a rati o of pol ymeri c fl avan-3-ol s to monomeri c
fl avan-3-ol s of <5 for Pi not Noi r grapes grown i n
Cal i forni a. The contri buti on of catechi ns to the sensa-
ti on of astri ngency and bi tterness i s di fferent from that
of pol ymeri c procyani di ns (25): the former was shown
to be more bi tter and l ess astri ngent than the l atter [see
al so a recent revi ew by Gawel (26)]. The di fference i n
tanni n-rel ated taste between Shi raz and Pi not Noi r
wi nes mi ght therefore be rel ated, at l east i n part, to the
di fference i n phenol i c composi ti on of thei r seeds. How-
ever, further evi dence i s needed to confi rm thi s.
ABBREVI ATI ONS USED
DP, degree of pol ymeri zati on; RP-HPLC, reverse
phase hi gh-performance l i qui d chromatography; G
1
,
monogal l ate; G
2
di gal l ate, etc.; LC-ESI /MS, l i qui d chro-
matography el ectrospray mass spectrometry; NMWCO,
nomi nal mol ecul ar wei ght cutoff; P, parent peak; P
1
,
procyani di n monomer (catechi n or epi catechi n); P
2
,
procyani di n di mer, etc.; PVPP, pol yvi nyl pol ypyrrol i -
done.
ACKNOWLEDGMENT
We thank Graham Jones of the Department of
Horti cul ture, Vi ti cul ture and Oenol ogy of the Uni versi ty
of Adel ai de for hel pful suggesti ons and Gayl e Bal dock
Table 1. Major m/z Signals Extracted fromPeak 13 by
LC-ESI/MS Analysis and Their Identities
m/z i denti ty
a
291 P1
b
443 P1G1
579 P2
731 P2G1
867 P3
1019 P3G1
1155 P4
1171 P3G2
1307 P4G1
1443 P5
1459 P4G2
1595 P5G1
1731 P6
1748 P5G2
1884 P6G1
2021 P7
2036 P6G2
2172 P7G1
2188 P6G3
2310 P8
2324 P7G2
2460 P8G1
2477 P7G3
2612 P8G2
2749 P9G1
2902 P9G2
a
Tentati ve assi gnment based on mol ecul ar wei ghts. Note that
al l speci es tabul ated were si ngl y charged.
b
Abbrevi ati ons: P,
procyani di n; P2, procyani di n di mer, etc.; G, gal l ate; G1, monogal -
l ate, etc.
30 J. Agric. Food Chem., Vol. 49, No. 1, 2001 Peng et al.
of the Austral i an Wi ne Research I nsti tute and Renata
Ri sti c of the Department of Horti cul ture, Vi ti cul ture and
Oenol ogy of the Uni versi ty of Adel ai de for techni cal
assi stance.
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Recei ved for revi ew May 31, 2000. Revi sed manuscri pt recei ved
October 18, 2000. Accepted October 19, 2000. The Grape and
Wi ne Research and Devel opment Corporati on of Austral i a
funded thi s work.
JF000670O
HPLC Analysis of Polymeric Tannins in Grape Seeds J. Agric. Food Chem., Vol. 49, No. 1, 2001 31