Analytica Chimica Acta 458 (2002) 15–27

Effect of oxygenation on polyphenol changes

occurring in the course of wine-making
Vessela Atanasova∗ , Hélène Fulcrand, Véronique Cheynier, Michel Moutounet
INRA-UMR Sciences pour l’Oenologie, 2 Place Viala, 34060 Montpellier, France
Received 25 June 2001; accepted 11 December 2001

Abstract
The influence of controlled oxygenation on the colour and phenolic composition of red wine was studied by UV–VIS
spectrophotometry, liquid chromatography (LC) coupled to diode array detection (DAD) and electrospray ionisation mass
spectrometry, and thiolysis. The comparison between the control and oxygenated wines demonstrated changes in colour
characteristics along with a significant increase in concentrations of pyranoanthocyanins, ethyl-bridged compounds and derived
pigments both with storage time and with oxidation. Principal component analysis was applied to wine analysis data measured
throughout the conservation period. The effect of the storage time and oxygenation was clearly reflected in this analysis.
Mass-spectrometric analysis of the wines demonstrated the presence of compounds which are markers of reactions
involving acetaldehyde. Two types of mechanisms were observed. The first concerns acetaldehyde condensation reac-
tions and the second, the cycloaddition process between anthocyanins and flavanols mediated by acetaldehyde, generating
tannin-pyranoanthocyanins.
The presence in wines of trimeric structures resulting from these mechanisms, as well as the results obtained after thiolysis
of the fraction containing polymeric species obtained by Fractogel chromatography, confirm that proanthocyanidins react
with acetaldehyde in the same way as flavanol monomers. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Wine; Oxygenation; Ageing; Pigments; Colour; Liquid chromatography; Liquid chromatography/electrospray ionisation mass
spectrometry

1. Introduction because they progressively react with other phenolic

Phenolic compounds play a major role in wine
quality. They contribute to sensory characteristics
of wines, in particular colour and astringency. Wine
colour (WC) is strongly influenced by grape compo-
sition and enological practices, such as wine-making
techniques, storage temperature, length of storage
[1,2] and oxygen exposure [3]. During conservation
and ageing of red wines, the concentration of grape
anthocyanins, initially responsible for WC, decreases
∗ Corresponding author. Fax: +33-4-99612683.
E-mail address: atanasov@ensam.inra.fr (V. Atanasova).
compounds, particularly flavanols [4,5]. This phe-
nomenon is thought to result in the colour change from
red-bluish of young wines towards the reddish-brown
colour of matured wines, as well as in the decrease
of wine astringency observed during ageing [6]. At
wine pH values, anthocyanins exist as two forms in
equilibrium, namely the red flavylium cation (A+ )
and the colourless hydrated hemiketal form (AOH),
the latter being predominant [7].
Various pathways involving both anthocyanin forms
have been proposed to explain the conversion of an-
thocyanins to new, more stable pigments [4,5,8]. The
first process is a direct reaction between flavanols (T)

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 6 1 7 - 8
16 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27
and anthocyanins (A) [4,5,9–11], leading to two kinds
of products, denoted A–T and T–A, according to the
position of the anthocyanin moiety. Recent work in
our laboratory [12] has revealed the presence in wine
of both types of compounds which, respectively arise
from addition of tannins onto A+ and from addition of
AOH onto the carbocation formed by acid-catalysed
cleavage of proanthocyanidin interflavanic bonds.
The second pathway concerns the reactions between
anthocyanins and flavanols mediated by acetaldehyde.
This mechanism, first described by Timberlake and
Bridle [8] and finally demonstrated by Fulcrand et al.
[13], starts with acetaldehyde condensation on fla-
vanols leading to a carbocation intermediate, which
reacts in turn with either another molecule of flavanol
or the hydrated form of an anthocyanin. Actually, the
detection of (+)-catechin ethyl-bridged dimers in wine
[14], of ethyl-bridged (epi)catechin dimers and trimers
and of (epi)catechin-ethyl-malvidin 3-glucoside in
model solutions [15] proved the occurrence of this
condensation reaction in red wines (Fig. 1).
The third type of reactions established in wines is
a cycloaddition process between anthocyanins and
some yeast metabolites possessing a polarisable dou-
ble bond. In particular, vinyl phenol [16], pyruvic acid
[17], acetaldehyde [18], were shown to react through
this mechanism, leading to more stable pigments,
Fig. 1. Structures of the pigments derived from the acetaldehyde-
mediated condensation between anthocyanins and flavanols.
R=flavanol units.
Fig. 2. Structures of pyranoanthocyanins. Pyrano-malvidin 3-gluco-
side: R=H; Carboxyl-pyrano-malvidin 3-glucoside: R=COOH;
(epi)catechin-pyrano-malvidin 3-glucoside: R = flavanol unit; fla-
vanol dimer-pyrano-malvidin 3-glucoside: R = flavanol dimer.
structurally allied to pyranoanthocyanins (Fig. 2). The
occurrence of these pigments has been demonstrated
in wine [16,19–22] and their remarkable stability
and resistance to sulfite bleaching has been estab-
lished [20,23]. Moreover, a similar structure resulting
from reaction of B2 (epicatechin-4␤-8-epicatechin)
procyanidin dimer, acetaldehyde and malvidin
3-glucoside in model solution has been recently pos-
tulated on the basis of MS data [24]. This structure
corresponds to a pyranoanthocyanin-tannin adduct,
the formation of which might be relevant to a new
stabilisation process of wine pigments.
All these reaction mechanisms may influence the
colour and colour stability of wine, as well as its gus-
tatory qualities related to the structure of the tannins.
Their relative importance, as well as the structure of
the end products, depends on the initial wine composi-
tion, but also on the presence of yeast metabolites and
on oxygen exposure. Thus, a mild oxygenation pro-
cess, referred to as micro-oxygenation, was proposed
to improve wine quality, assuming that orientation of
phenolic compound reactions towards the oxidative
ways resulted in more coloured and less astringent
products [3,25]. Among the reaction pathways de-
scribed above, only those involving acetaldehyde, i.e.
formation of pyranoanthocyanins and of ethyl-bridged
adducts, are expected to be favoured by the presence
of oxygen. Acetaldehyde is a natural compound oc-
curring in wines, produced either by yeast metabolism
during fermentation [26] or by ethanol oxidation in
presence of phenolic compounds [27].
 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27
The purpose of the present work is therefore, to
evaluate the effect of oxygenation on red WC and
phenolic composition and, to elucidate possible rela-
tionships between the formation of various types of
derived anthocyanins and WC changes.
2. Experimental
2.1. Wine sample
The wine analysed was a blended red wine, made
from Vitis vinifera var. Cabernet Sauvignon (60%) and
Tannat (40%), vintage 1999. The wine was distributed
into six tanks; three were saturated with N2 (control)
and three were dispensed continuously with a flow
providing 5 ml l−1 pure oxygen through a microdif-
fuser (oxygenated). Thus, the experiment (including
storage in tanks, fractionation and analyses) was per-
formed in triplicate.
2.2. Fractionation procedure
A 2 ml of the wine was fractionated on a Toyopearl
TSK gel HW-50 (F) column (120 mm × 12 mm,
1 ml min−1 ). Elution was monitored by absorbance
at 280 nm. The first elution performed with a sol-
vent containing ethanol/water/trifluoroacetic acid
(55:45:0.005, v/v/v) was stopped just after the fla-
vanol monomers were eluted. This fraction (F1) con-
tains mainly monomeric compounds such as phenolic
acids, anthocyanins, flavonols and (epi)catechin. The
polymeric fraction (F2) was eluted with 50 ml of ace-
tone/water (60:40, v/v). F1 and F2 were taken to dry-
ness under vacuum and dissolved in 2 ml of methanol.
2.3. Liquid chromatography/diode
array detection (LC/DAD)
LC/DAD analyses were performed using a Waters
2690 system equipped with an autosampler sys-
tem, a Waters 996 photodiode array detector and
Millenium 32 chromatography manager software.
Separation was achieved on a Lichrospher 100-RP18
column (5 ␮m packing, 250 mm × 4 mm i.d.) pro-
tected with a guard column of the same material
(Merck, Darmstadt, Germany). The elution conditions
were as follows: 1 ml min−1 flow rate; oven tempera-
17
ture, 30 ◦ C; solvent A, water/formic acid (95:5, v/v);
solvent B, acetonitrile/water/formic acid (80:15:5,
v/v/v), elution began with isocratic A/3% B for 7 min,
from 3 to 20% B in 15 min, from 20 to 30% B in
8 min, from 30 to 40% B in 10 min, from 40 to 50%
B in 5 min and from 50 to 80% B in 5 min, followed
by washing and re-equilibrating the column. UV–VIS
spectra were recorded from 220 to 600 nm. The an-
thocyanins were quantified by using an external mal-
vidin 3-glucoside chloride standard (isolated in our
laboratory). Peak areas at 520 nm were converted to
milligram per liter equivalent malvidin 3-glucoside
using a standard curve prepared by different con-
centrations of malvidin 3-glucoside chloride in 2%
HCl.
2.4. Thiolysis reaction and LC analyses
Equal volumes of fraction F2 and thiolytic reagent
(toluene-␣-thiol 5% in 0.2 M HCl in methanol) were
mixed. After sealing, reaction was carried out at 60 ◦ C
for 5 h, then the vials were cooled and the reaction
products analysed directly by LC.
The LC apparatus was a Kontron system (Milan,
Italy) equipped with a 460 autosampler, a 325 pump
system, a 430 detector, and an 450 MT2 data system.
The column was a Nucleosil C18 (3 ␮m packing,
125 mm × 4 mm i.d., Dûren, Germany). The elution
conditions were: 0.8 ml min−1 flow rate; oven temper-
ature, 30 ◦ C; solvent A, water/formic acid (98:2, v/v);
solvent B, acetonitrile/water/formic acid (80:18:2,
v/v/v), elution began with isocratic A/15% B for 5 min,
followed by linear gradients from 15 to 75% B in
15 min and from 75 to 100% B in 5 min and isocratic
elution with 100% B for 2 min. Detection was mon-
itored at 280 nm. Identification of flavanol monomers
and of the corresponding benzylthioethers was based
on both standard retention times and NMR/MS char-
acterisations established earlier [28–30]. Mass spec-
trometric (MS) detection is described below.
2.5. Colour measurement
Absorbance measurements were made with a
Uvicon 930 spectrophotometer, and were converted
to absorbance A with 10 mm light path and corrected
for the dilution. The wine samples were filtered
(0.45 ␮m) before being analysed.
18 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27
Colour density (CD) was defined as the sum of
absorbances at 620, 520 and 420 nm [31] and tint (T)
as the ratio between the absorbances at 420 and 520 nm
[32], with absorbance measurements performed di-
rectly on the wine, using a 1 mm pathlength cell.
WC, total colour of pigments (WCP), colour due
to derivatives resistant to SO2 bleaching (CDR SO2 ),
colour due to co-pigmentation (CA), were then deter-
mined using a method adapted from that of Boulton
et al. [33] and all measurements were made at 520 nm.
WCP: The wine was diluted in 2% HCl so as to
obtain an absorbance at 520 nm of ca. 0.1–0.3 (Awine )
in a 10 mm pathlength cell. Absorbance was measured
after 3–4 h to ensure complete conversion of antho-
cyanins into the flavylium form.
WC: A 50 ␮l of 12% (v/v) acetaldehyde solution
was added to 5 ml of wine to release anthocyanins
eventually involved in bisulfite adducts and ab-
sorbance was measured after 45 min (Aacet ) in a 1 mm
pathlength cell.
CA: The acetaldehyde-treated wine (Aacet ) was
diluted in a solution of 12% ethanol and potas-
sium bitartrate at wine pH to dissociate eventual
copigment-anthocyanin complexes. The dilution was
adapted so as to obtain an absorbance value at 520 nm
of ca. 0.1–0.3 (Anoncopig ) in a 10 mm pathlength cell.
CA was then calculated as: CA = Aacet − Anoncopig .
CDR SO2 : A 75 ␮l of 20% (w/v) sodium metabisul-
fite solution was added to 5 ml of wine, and the ab-
sorbance measured after 10 min (ASO2 ) in a 1 mm
pathlength cell.
Chemical age of wine (CAW) was calculated as
proposed by Somers and Evans [34]: CAW (%) =
100 × (ASO2 /Aacet ).
Based on the results obtained above, the colour due
to pigments non-resistant to SO2 bleaching (CPNR
SO2 ) and colour due to derived pigments (CDP) were
calculated as follows. CPNR SO2 was determined as
the difference between WCP and CDR SO2 .
Since, free anthocyanins (FA) contribute to a large
extent to CPNR SO2 , the colour due to derivatives
non-resistant to SO2 bleaching (CDNR SO2 ) was
then calculated by subtracting the concentration of
FA from CPNR SO2 . In this calculation, the amount
of FA in mg l−1 equivalent malvidin 3-glucoside
chloride (molecular mass 529) determined by LC was
converted to absorbance A using the molar absorptiv-
ity value (ε) of 26051 l mol−1 cm−1 determined with
malvidin 3-glucoside isolated in our laboratory.
Hence, A = C (mg l−1 )/20.3.
CDNR SO2 = WCP − CDR SO2 − FA
Thus, the CDP represents the sum of CDR SO2 and
CDNR SO2 .
CDP = WCP − FA = CDR SO2 + CDNR SO2
2.6. MS apparatus and LC/MS analyses
MS analyses were performed on a Sciex API I
Plus simple quadrupole mass spectrometer including
an electrospray ion source (Sciex, Thornhill, On-
tario, Canada). The mass spectrometer was operated
in the positive and negative ion modes, in the range
280–1500 amu (in negative ion mode 280–1200 amu).
The ion spray voltage was +5600 V (−4000 V) and
the orifice voltage +64 V (−70 V). Chromatographic
separation was achieved using an ABI 140B solvent
delivery system (Applied Biosystems, Weiterstadt,
Germany), with the same solvents as above at a flow
rate of 280 ␮l min−1 . The column was a LiChro-
spher 100-RP18 (5 ␮m, 250 mm × 2 mm i.d.; Merck,
Darmstadt, Germany). The elution conditions were as
follows. (1) For the non-thiolysed samples: isocratic
conditions with 3% B (see above) for 4 min, linear
gradients from 3 to 15% B in 11 min, from 15 to 50%
B in 30 min and from 50 to 100% B in 5 min, followed
by washing and re-equilibrating the column. (2) For
the thiolysed samples: linear gradients from 3 to 15%
B in 2 min, from 15 to 50% B in 34 min and from
50 to 100% B in 10 min, followed by washing and
re-equilibrating the column. The oven temperature
was 30 ◦ C. The column was connected to the elec-
trospray interface via a fused silica capillary (length
100 cm, 75 ␮m i.d.). The sample was injected with
a rotary valve (Rheodyne model 8125) fitted with a
20 ␮l sample loop. The absorbance at 280 nm was
monitored by an ABI 785A programmable absorbance
detector. The flow was split after UV detection so that
70 ␮l min−1 went to the electrospray source.
2.7. Statistical data treatment
Principal component analysis was performed using
StatBox (c) 1995–1997, Grimmer Logiciels Version
2.5, Grimmersoft, Paris, France.
 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27 19

Fig. 3. Liquid chromatogram at 520 nm of oxygenated wine at 7 months of storage. (1) Dp3g, (2) Cy3g, (3) Pt3g, (4) Pn3g, (5) Mv3g,
(1 ) Dp3g-ac, (2 ) Cy3g-ac, (3 ) Pt3g-ac, (4 ) Pn3g-ac, (5 ) Mv3g-ac, (1 ) Dp3g-coum, (2 ) Cy3g-coum, (3 ) Pt3g-coum, (4 ) Pn3g-coum,
(5 ) Mv3g-coum, (6) Carboxyl-pyrano-Dp3g, (7) Carboxyl-pyrano-Pt3g, (8) Carboxyl-pyrano-Mv3g, (9) Carboxyl-pyrano-Mv3g-ac, (10)
Carboxyl-pyrano-Mv3g-coum, (11) cat-ethyl-Mv3g and (12) epicat-ethyl-Mv3g.

3. Results and discussion
The anthocyanin composition of the wine sample
was established by LC/DAD and LC/ESI-MS anal-
yses. The liquid chromatogram recorded at 520 nm
for the oxygenated wine sample at 7 months time
is presented in Fig. 3. The chromatogram shows
the presence of 15 anthocyanins, originating from
grapes. Peaks 1–5 correspond to the 3-glucosides
of delphinidin, cyanidin, petunidin, peonidin and
malvidin. Peaks 1 –5 were identified as the corre-
sponding 3-acetylglucosides and peaks 1 –5 as the
3-p-coumaroylglucosides. In addition to the native
grape pigments, the chromatogram shows the pres-
ence of a series composed of peaks 6, 7, 8, 9 and 10,
showing UV–VIS spectra characteristic of pyranoan-
thocyanins. These molecules detected at m/z 531,
545, 559, 601 and 705 in the negative ion mode, re-
spectively, were identified to the pyranoanthocyanin
adducts resulting from reaction of pyruvic acid with
delphinidin 3-glucoside (carboxyl-pyrano-Dp3g), pet-
unidin 3-glucoside (carboxyl-pyrano-Pt3g), malvidin
3-glucoside (carboxyl-pyrano-Mv3g), malvidin 3-ace-
tylglucoside (carboxyl-pyrano-Mv3g-ac) and mal-
vidin 3-p-coumaroyglucoside (carboxyl-pyrano-Mv
3g-coum) [17,18].
LC/DAD-MS analysis of the wine in the positive
ion mode allowed a signal at m/z 517 to be detected
(Fig. 4). This signal was attributed to the cycloadduct
of Mv3g with acetaldehyde (pyrano-Mv3g), by com-
parison with the values (retention time and spectrum)
reported by Benabdeljalil et al. [18] for this pyra-
noanthocyanin. Interpretation of this mass signal was
confirmed by detection of a fragment ion at m/z 355
attributed to the aglycone moiety after the loss of
glucose. In Fig. 3, pyrano-Mv3g corresponds to the
shoulder on Cy3g-ac. Occurrence of this compound in
wines led us to search allied products resulting from
20 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27
Fig. 4. Mass chromatograms extracted from the TIC from oxygena-
ted wine at t7months . The m/z values at 517.805 and 1093 corre-
spond, respectively to pyrano-Mv3g, (epi)catechin-pyrano-Mv3g
and flavanol dimer-pyrano-Mv3g.
ethanal cycloaddition between Mv3g and flavanol
polymers (Fig. 2). This structure was first postulated
by Francia-Aricha et al. [24] on the basis of the
UV–VIS and mass spectra of a product formed by
reaction of procyanidin B2, Mv3g and acetaldehyde
in a model wine solution. The mass chromatogram
extracted at m/z 805 and 1093 from the total ion cur-
rent (TIC) of the wine (Fig. 4) allowed us to detect
the pigments corresponding to the structure shown
in Fig. 2 ((epi)catechin-pyrano-Mv3g and flavanol
dimer-pyrano-Mv3g, respectively). The UV spectra of
these compounds have a maximum wavelength in the
visible region at 495 nm (Fig. 5) and are very similar
to that published by Francia-Aricha et al. [24].
As for the condensation process between antho-
cyanins and flavanols mediated by acetaldehyde,
LC/DAD-MS analysis of the wine conducted in the
positive ion mode showed the presence of peaks
detected at m/z 781, 795, 779 and 809 (Fig. 6). These
signals were attributed to the ethyl-bridged pigments
of, respectively Dp3g, Pt3g, Pn3g and Mv3g with
(epi)catechin. The Mv3g derivatives (peaks 11 and 12
in Fig. 3) were the only compounds detected on the
Fig. 5. UV spectra of the pigments detected at m/z 805 and 809.
LC/DAD profile, the other ethyl-bridged pigments be-
ing coeluted with other wine pigments. Their absorp-
tion maxima were at 545 nm (Fig. 5), in agreement
with earlier work [15,35]. Regarding the structures of
these compounds (Fig. 1), the presence of an addi-
tional asymmetric carbon within the ethyl bridge and
of two linkage sites (C6, C8) for each flavanol epimer
(catechin, epicatechin) explained that several peaks,
corresponding to the different possible stereo—(R, S)
and regio—(C8–C8, C8–C6) isomers, were detected
in the mass chromatogram (Fig. 6). Besides, one new
trimeric compound was found in the TIC trace of
wine. Its molecular ion peak detected at m/z 1097
corresponds to the Mv3g-ethyl-flavanol dimer struc-
ture, showing for the first time the occurrence of such
oligomers in wine.
The presence of the carboxyl-pyranoanthocyanins
(Pyran), pyranoanthocyanins, flavanol-pyranoantho-
cyanins and of anthocyanin-ethyl-flavanol adducts
in wine were therefore, confirmed both by UV–VIS
and mass detection, following LC separation. This
indicates that a great diversity of products can be
generated during wine ageing, their respective lev-
els depending on the nature and relative amounts of
flavanol and anthocyanin molecules as well as on
acetaldehyde availability.
In order to obtain further insight into the occurrence
of polymeric compounds derived from anthocyanin
and tannin reactions, the wine was fractionated on a
Toyopearl column to separate monomeric and dimeric
phenols (recovered in F1) from higher molecular mate-
rial (eluted in F2). In fraction F1, the same monomeric
and dimeric pigments as found in wine were detected,
confirming that lower molecular weight derivatives
were eluted in this fraction [12]. The results obtained
 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27
Fig. 6. Mass chromatograms extracted from the TIC from oxyg-
enated wine at t7months . The m/z values at 781, 795, 809 and 1097
correspond, respectively to Dp3g-ethy-(epi)catechin, Pt3g-ethyl-
(epi)catechin, Pn3g-ethyl-(epi)catechin, Mv3g-ethyl-(epi)catechin
and Mv3g-ethyl-flavanol dimer.
from direct analysis of F2 did not provide additional
information on possible products yielded by tannin
and anthocyanin reactions. Therefore, LC-MS analy-
sis in the positive ion mode was performed after thiol-
ysis of F2 to detect eventual derivatives arising from
derived polymeric species. This alternative approach
allows the depolymerisation of proanthocyanidins, in
the presence of toluene-␣-thiol, into their constituent
21
Fig. 7. Mass chromatograms extracted from the TIC from oxy-
genated wine polymeric fraction. The m/z values at 927 and
643 correspond, respectively to benzylthioether derivative of
(epi)catechin-pyrano-Mv3g and Mv3g-ethyl benzylthioether.
units. In fact, cleavage of the interflavanic bonds con-
necting proanthocyanidin units releases terminal units
as unchanged flavan-3-ols and extension units as the
corresponding benzylthioether derivatives, allowing
the determination of the respective amounts of each
constitutive unit and of the mean degree of polymeri-
sation (mDP) [28–30]. Thiolysis can also be of use to
characterise derived tannins formed in the course
of wine-making, again allowing one to discriminate
between terminal and other units [12].
Two new types of benzylthioether compounds were
detected in the TIC trace of thiolysed F2 fraction at
m/z 927 and 643, along with the signals correspond-
ing to genuine proanthocyanidin units (Fig. 7). In
addition, the similarity of their absorbance spectra
(Fig. 8) with the compounds detected, respectively
at m/z 805 and 809 suggest that they are the ben-
zylthioether derivatives of (epi)cat-pyrano-Mv3g and
Mv3g-ethyl benzylthioether, respectively (Fig. 9).
These hypotheses were confirmed by the presence
of ion peak fragments at m/z 803 (Fig. 10a) and 519
(Fig. 10b) corresponding to the loss of a thioether
residue (124 amu) from each molecule.
The mass corresponding to (epi)catechin-ethyl ben-
zylthioether derivative (m/z 441) was not detected,
indicating, on the one hand, the absence of products
22 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27

Fig. 8. UV spectra of the pigments detected at m/z 927 and 643.

resulting from acetaldehyde-induced polymerisation

of flavanols, and on the other hand, that cleavage of the
molecule primarily concerns the (epi)catechin-ethyl
bridge. This confirms that this bridge is more sensi-
tive to thiolysis than that between the ethyl group and
Mv3g as shown earlier [36]. The results presented
above thus, indicate that the oligomeric or polymeric Fig. 10. Mass spectra obtained from TIC chromatogram at 45.1
tannins are involved in acetaldehyde induced react- and 48.6 (a) and at 41.6 (b). Characteristic fragments (m/z 803
ions, which yield both tannin-ethyl-anthocyanins and and 519) were observed.
tannin-pyranoanthocyanins.
Quantification of the pigments resulting from Table 1 shows the influence of storage time and
acetaldehyde reactions (ethyl and pyrano products), as oxygenation on the pigment pool, as determined from
well as their benzylthioether derivatives, requires fur- LC and spectral data for the studied wines. Analyses
ther investigation in order to evaluate their importance have been run at three different times of storage, 0
in wine qualities with respect to micro-oxygenation. month (t0 ), 1 month (t1 ) and 7 months (t7 ).

Fig. 9. Structures of benzylthioether derivative of (epi)catechin-pyrano-Mv3g (a) and Mv3g-ethyl benzylthioether (b).
 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27 23

Table 1
Phenolic compounds and colour characteristics of wines
Compounds 0 Month 1 Month 7 Months

Wine Control wine Oxygenated wine Control wine Oxygenated wine

CD (A.U.)a 21.8 ± 0.2 a 19.9 ± 0.4 b 20.7 ± 0.9 b 18.5 ± 0.1 c 21.1 ± 0.1 b
Ta 0.2 ± 0a 0.5 ± 0b 0.5 ± 0b 0.6 ± 0c 0.6 ± 0c
WC (A.U.)a 21.30 ± 0.1 a 13.87 ± 0.25 b 14.17 ± 0.12 b 11.93 ± 0.21 c 12.50 ± 0d
CAW (%)a 19 ± 0a 28.67 ± 2.31 b 30.00 ± 0b 39.33 ± 0.58 c 42.67 ± 0.58 d
FA (A.U.)b 23.20 ± 0.70 a 21.64 ± 0.13 b 22.04 ± 1.03 b 14.92 ± 0.27 c 12.62 ± 0.27 d
Pyran (mg l−1 )b 17.27 ± 0.28 a 16.99 ± 0.54 a 17.82 ± 0.70 a 19.47 ± 0.47 b 21.04 ± 0.45 c
Ethyl-bridged compounds (mg l−1 )b 2.22 ± 0.18 ac 2.00 ± 0.07 b 2.25 ± 0.16 c 1.83 ± 0.14 b 2.33 ± 0.09 c
mDPb 10.1 ± 0.4 a 10.1 ± 0.7 a 11.0 ± 0.9 a 12.6 ± 0.3 b 12.2 ± 0.9 b
Native tannins (mg l−1 )b 1434 ± 50 ab 1382 ± 33 a 1455 ± 25 b 1340 ± 44 a 1214 ± 39 c
CA (A.U.)a 9.10 ± 0.7 a 4.20 ± 0.26 b 4.33 ± 0.21 b 2.67 ± 0.15 c 2.43 ± 0.06 d
WCP (A.U.)a 41.77 ± 0.61 a 39.27 ± 0.90 b 39.27 ± 0.15 b 29.17 ± 0.07 c 28.87 ± 0.40 c
CDR SO2 (A.U.)a,b 4.03 ± 0.06 a 3.93 ± 0.38 a 4.20 ± 0.01 a 4.73 ± 0.12 b 5.33 ± 0.06 c
CDNR SO2 (A.U.)a,b 14.53 ± 1.14 a 13.69 ± 0.48 a 13.03 ± 0.91 a 9.52 ± 0.74 b 10.91 ± 0.65 c
CDP (A.U.)a 18.57 ± 1.20 a 17.62 ± 0.85 a 17.23 ± 0.91 a 14.25 ± 0.69 b 16.25 ± 0.65 c
A.U.: absorbance units.
a Spectrophotometric method.
b LC method.

The name letters indicate no significant difference between the corresponding values.

The concentration of FA significantly dropped dur-
ing storage, as expected from earlier work [5,37].
However, it can be noted that their loss was signif-
icantly higher in the oxygenated wines than in the
control ones after 7 months. Pyran gradually accumu-
lated in both types of wines (control and oxygenated)
whilst the amount of anthocyanin-ethyl-(epi)catechin
decreased with storage time, but less in oxygenated
wines. This confirms that addition of pyruvic acid
to anthocyanins is not influenced by oxygen, as ex-
pected, and suggests that is a slow process, since both
precursors are present in wine at the end of fermen-
tation [38]. In contrast, changes in anthocyanin-ethyl-
(epi)catechin content reflect the instability of these
compounds [39], and confirm that their formation
is favoured by oxygen. In fact, formation of these
derivatives slightly exceeded their degradation in the
oxygenated wine.
The mDP of wine proanthocyanidins, calculated as
the ratio between the total number of units and the
number of terminal units, increased with time whilst
the total amount of proanthocyanidin units released
by thiolysis diminished in the oxygenated wines at
t7months . The observed changes are essentially due to
losses of terminal units, suggesting that the reactions
modifying polymers preferentially involved the ter-
minal units.
Among the derived pigments which can be formed
in wine processing, only a few have been identified
up to now and can be individually assayed by LC.
Consequently, a combination of spectrophotometric
and LC methods was used to quantify and charac-
terise native and derived pigments, as described in the
experimental section. Statistical analysis of the mea-
sured values indicated that significant changes took
place throughout the storage period and as a result of
7-month oxygen exposure.
WC and WCP, corresponding, respectively to the
WC (i.e. absorbance measured at wine pH after de-
combining anthocyanin bisulfite adducts) and the total
WC measured after converting colourless hemiketal
anthocyanins and bisulfite adducts into the corre-
sponding flavylium pigments, decreased significantly
with time. This decrease was concomitant with the
loss of FA, meaning that formation of anthocyanin
derived pigments did not compensate for free antho-
cyanin degradation. Moreover, the decrease of WC
was slightly less important in oxygenated wines than
in the corresponding control wines, in spite of the
higher free anthocyanin degradation rate, suggesting
24 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27
that oxidative reactions generated more pigments than
non oxidative ones. WCP values were not influenced
by oxygen exposure, indicating that the total amount
of pigments was identical in oxygenated and control
wines but that a larger proportion was present under
colourless hydrated form (AOH) in the latter.
CD, representing the sum of absorbances at 420,
520 and 620 nm, also diminished with time, but less
in oxygenated wines, in agreement with literature
data [40]. This is usually ascribed to the formation of
derived pigments from genuine anthocyanins. In par-
ticular, formation of ethyl-bridge anthocyanin-tannin
adducts is likely to contribute to the increase in
CD values as the absorbance of these molecules
at 420 and 620 nm relative to that measured at
520 nm is higher than that of genuine anthocyanins
[8,36,39]. This hypothesis is supported by the fact
that anthocyanin-ethyl-(epi)catechin concentration
evolved in the same way as CD. These molecules were
also shown to be present only under the flavylium
form at wine pH values [39]. Pyranoanthocyanins
may also participate in the higher CD of oxygenated
wines as they show higher 420 nm absorbance values
than anthocyanins [18,20] and their formation should
be favoured by oxidation.
CAW, corresponding to the ratio between pig-
ments resistant to sulfite bleaching, WC and T, cor-
responding to the ratio between the absorbances at
420 and 520 nm, increased with time and with oxy-
genation for CAW, as observed earlier [5]. This is
classically attributed to conversion of anthocyanins to
non-discolourable pigments. Formation of pyranoan-
thocyanins is probably associated with these changes
since they are resistant to sulfite bleaching and absorbs
in the range 400–520 nm, with maximum absorbance
around 500 nm. Ethyl-bridged tannin-anthocyanin
adducts which have been shown to resist hydration,
should also be resistant to sulfite addition and may
contribute to the observed changes. Moreover, the T
value measured for these pigments is higher than that
of genuine anthocyanins.
Colour due to the derived pigments (CDP) was
calculated as the sum of CDR SO2 and CDNR SO2
bleaching. The latter correspond mainly to molecules
having their C-4 position non-substituted, including
native anthocyanins and part of derived pigments.
Their contribution to colour, which is referred to as
CDNR SO2 in Table 1, can be calculated by subtract-
ing the CDR SO2 value and the colour due to FA from
WCP. The percentage of CDP with respect to WCP
increased with time storage, and was significantly
higher in the oxygenated wines.
All these results confirm that formation of polyme-
ric derivatives takes place during wine ageing [5,34],
but it is significantly favoured by oxygenation [40,41].
Further stabilisation of WC is due to co-pigmen-
tation, which consists of displacement of the antho-
cyanin hydration equilibrium towards the coloured
flavylium form, as a result of interaction (␲-␲ stack-
ing) between its planar structure and that of other
molecules referred to as co-pigments [42].
The CA significantly diminished with storage time
and was lower in oxygenated wines than in control
wines at t7months . This decrease follows the free antho-
cyanin concentration, suggesting that co-pigmentation
does not involve so much new pigments. This finding
agrees with the structure of most derived new pig-
ments identified until now, and especially of those
accumulating in oxygenated wines (i.e. pyranoan-
thocyanins, (epi)catechin-ethyl-Mv3g), which are
resistant to hydration [8,23,39].
3.1. Principal component analysis
PCA was applied to the 15 wine samples taken over
storage time (0, 1 and 7 months) based on the 14
parameters reported in Table 1. Four components have
an eigenvalues higher than 1. The first two principal
components retained 85% of the variance.
Fig. 11a shows the contribution of the variables to
the first two axes. The first axis, representing 69%
of the total variance, contrasted concentrations of FA
and of tannins, WCP, CDNR SO2 , CA, CDP and WC
with Pyran content, CDR SO2 , CAW, and T. Tannins,
FA, WCP, and CDNR SO2 were highly correlated
between them, and to a lesser extent to CDP, WC and
CA Carboxyl-pyranoanthocyanin content, colour due
to derivatives resistant to SO2 bleaching, CAW and
T were positively correlated between them and neg-
atively correlated with FA, WCP, CDNR SO2 , CA,
CDP and WC.
Ethyl-bridged pigments, and CD were correlated,
and contributed to the second axis (16% of the total
variance) along with CDR SO2 and Pyran. The pos-
itive correlation between ethyl-bridged pigments and
CD, and the lack of correlation between CD and both
 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27 25

Fig. 11. Contribution of the variables (a) and distribution of wines (b) in the two-dimensional coordinate system defined by the first two
principal components: (䉱)control wines t0month , (䊏) control wines t1month , (䊐) oxygenated wines t1month , (䊉) control wines t7months , (䊊)
oxygenated wines t7months .

T and WC suggest that the increase in CD is mostly at-
tributable to that of absorbance at 620 nm, which may
reflect the bathochromic shift in the visible spectrum
of ethyl-bridged pigments compared to that of genuine
anthocyanins.
Fig. 11b shows the distribution of wine samples
in the plane defined by the first two components.
Four groups, distinguishing the wines as a function
of the storage time and effect of oxygenation, can be
observed.
Control and oxygenated samples were still grouped
together after one month of ageing, but they were
shifted to the left of t0 wines. This means that time
was a prominent factor with respect to oxygenation
for the first few weeks of the experiment. After 7
months of storage, the dispersion of two groups of
26 V. Atanasova et al. / Analytica Chimica Acta 458 (2002) 15–27

wines was remarkable, meaning that the control and changes. However, they might be the witness of simi-
the oxygenated wines took on different characteristics. lar oligomeric and polymeric derivatives arising from
Wines are clearly separated in function of their the same reaction mechanisms.
conservation time along the first axis, which can thus
be interpreted as a “maturation” axis, with ageing
4. Conclusions
increasing from right to left. Young wines were
characterised by higher levels of FA, tannins, WC,
Statistical analysis of the measured values indi-
WCP, CA, but also of derivatives discoloured by SO2
cated that significant changes took place throughout
(CDNR SO2 ). Location of total derived pigments
the storage time and as a result of 7-month oxygen
(CDP) in the same region can be explained by the fact
exposure. The wines at t0month and t1month were char-
that CDNR SO2 represent between 67% and 76% of
acterised by higher levels of pigments non-resistant
colour due to derived pigment over the storage time
to SO2 bleaching, including in particular genuine
(Table 1). It is interesting to note that pyranoantho-
anthocyanins. With time, their concentration progres-
cyanins, CDR SO2 , CAW, mDP and T were features
sively diminished and more stable structures such
associated with longer storage time. This indicates
as pyranoanthocyanins gradually accumulated. Oxy-
that pyranoanthocyanins and other pigments resistant
gen exposure enhanced anthocyanin degradation and
to sulfite bleaching accumulated during wine ageing
favoured reactions involving acetaldehyde leading
while the FA content decreased. Conversion of gen-
to ethyl-bridged compounds. Both processes may
uine anthocyanins into these derived pigments was
partly explain the change in colour characteristics and
concomitant with an increase of T values, the older
decrease of astringency.
wines showing a more tawny colour than the young
Detection of other molecular species, including
ones, as classically observed.
[(epi)catechin]n -pyranoanthocyanin, with n = 1 and
Oxygenated and control wines were separated along
2, anthocyanin-ethyl-(epi)catechin adduct, and benzyl-
the second axis, which can thus be interpreted as an
thioethers arising from thiolysis of proanthocyanidin-
oxygenation axis. Oxygenated wines appeared to be
pyranoanthocynain, and of proanthocyanidin-ethyl-
associated with higher values of CD and ethyl-bridged
malvidin 3-glucoside indicate that proanthocyanidins,
pigment concentrations, suggesting that formation
as well as flavanol monomers, are involved in both
of ethyl-bridged pigments is indicative of oxidation
types of acetaldehyde-induced reactions. Conversion
reactions, and contributes to CD changes. This is
to pyranoanthocyanins may participate in the degra-
in agreement on the one hand, with their formation
dation of ethyl-linked pigments. One of the possible
mechanism involving acetaldehyde, which is formed
mechanisms involves cleavage of the ethyl-anthocyanin
in larger amounts under oxidative conditions [27],
bridge proposed by Escribano-Bailon [39], then follo-
on the other hand, with their characteristic UV–VIS
wed by addition of the resulting vinyl-proanthocyanidin
spectrum [8].
onto the anthocyanin moiety. The vinyl-(epi)catechin
The results of PCA of LC and spectrophotometric
intermediates have been detected earlier in solutions
data suggest that the new pigments generated dur-
containing acetaldehyde and (epi)catechin [43].
ing wine ageing showed higher T values and were
more resistant to sulfite bleaching than anthocyanins
whereas 7-month oxygenation induced the formation Acknowledgements
of purple pigments. After 7-month ageing, control
and oxygenated wines were characterised by higher Thanks are due to Society Oenodev for providing
levels of Pyran and of (epi)catechin-ethyl-malvidin the wine samples.
3-glucoside, respectively. Although, the characteris-
tics (UV–VIS spectra, resistance to sulfite bleaching)
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