Identification by mass spectrometry of new compounds arising from the reactions involving malvidin-3-glucoside-(O)-catechin, catechin and malvidin-3-glucoside

Article abstract of DOI:10.1002/rcm.6330

RATIONALE The aim of this work was to study the putative reactions that could occur in red wines between malvidin-3-glucoside-(O)-catechin (mv3glc-(O)-cat) adduct and catechin (cat) or malvidin-3-glucoside (mv3glc) in presence of acetaldehyde. METHODS Mv3

Full text of DOI:10.1002/rcm.6330

Research Article
Received: 21 May 2012
Accepted: 21 June 2012
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 2123–2130
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6330
Identification by mass spectrometry of new compounds arising
from the reactions involving malvidin-3-glucoside-(O)-catechin,
catechin and malvidin-3-glucoside
*
Luís Cruz, Nuno Mateus and Victor de Freitas
Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto,
Rua do Campo Alegre, 687, 4169-007 Porto, Portugal
RATIONALE: The aim of this work was to study the putative reactions that could occur in red wines between malvidin-3-
glucoside-(O)-catechin (mv3glc-(O)-cat) adduct and catechin (cat) or malvidin-3-glucoside (mv3glc) in presence of acetaldehyde.
METHODS: Mv3glc-(O)-cat adduct (1 mM) was incubated with catechin (or mv3glc) in the presence of acetaldehyde
(molar ratio of 1:4:10) in 12% ethanol/water at pH 3.2, protected from light and placed in the oven at 30 ꢀC. The
formation of the new compounds was monitored by liquid chromatography-diode-array detection/electrospray ionization
mass spectrometry (LC-DAD/ESI-MS) analysis in the positive and negative ion mode.
RESULTS: The LC-DAD/ESI-MS characterization allowed the confirmation of the structures of the methylmethine-linked
cat-mv3glc-(O)-cat ([M–H]– m/z 1097) and mv3glc-mv3glc-(O)-cat ([M]+ m/z 1301) adducts.
CONCLUSIONS: The studies performed in model solutions showed that the colorless mv3glc-(O)-cat adduct can
undergo some of the characteristic reactions of anthocyanins and flavan-3-ols that occur in red wine in the presence of
acetaldehyde forming new methylmethine-bridged compounds. Copyright © 2012 John Wiley & Sons, Ltd.
Red wine is a complex matrix where polyphenolic compounds
such as anthocyanins and flavan-3-ols (e.g. catechins and
procyanidins) play the major role in its organoleptic properties
(color and flavor). However, these compounds are very suscep-
tible to undergo transformations during processing and
storage. Therefore, several chemical reactions take place
between anthocyanins, flavan-3-ols and also with small mole-
cules (pyruvic acid, acetaldehyde, acetoacetic acid, etc.) yielding
new stable anthocyanin-derived pigments (e.g. pyranoantho-
cyanins like vitisin A, vitisin B, methylpyranoanthocyanin,
etc.) which contribute to the modification of the sensorial
properties of red wines.[1–3] Among the several reactions invol-
ving red wine polyphenols, direct and acetaldehyde-mediated
condensations between anthocyanins and flavan-3-ols have
been widely studied.[4–12] One of the first reactions described
in red wines was the polymerization reaction between antho-
cyanins and flavan-3-ols mediated by acetaldehyde,[4,5,7,13]
which is essentially a by-product of ethanol oxidation.[14]
Acetaldehyde could also arise from yeast metabolism during
alcoholic fermentation. This kind of reaction leads to the forma-
tion of the purple-red anthocyanin-flavan-3-ol pigments linked
by methylmethine bridges (being their lmax is batochromati-
cally shifted from genuine anthocyanins).
and formation pathway of F-A adducts in red wines is well
documented in the literature and seems to be coherent among
the authors, in opposition to the case of A-F pigments. In fact,
the formation of A-F adducts in red wines is described in the
literature through a mechanism in which a nucleophilic attack
of flavanols (C-6/C-8) occurs towards the electropositive C-4
of anthocyanin giving rise to a colorless product (flavene
structure). This adduct could further evolve to the colorless
bicyclic form (additional interflavanolic linkage type-A,
A-(O)-F) or undergo oxidation to give the red pigment A+-F,
which could dehydrate to the orange-yellow xanthylium
salt.[10,12,15–17] The formation of a colorless flavene-type struc-
ture in red wines was described for the first time by Jurd in
1967.[18] The flavene form was later detected in model solutions
by UV–vis and mass spectrometry.[19] Besides this adduct, the
xanthylium form was also detected by UV–vis spectro-
scopy.[20,21] The bicyclic form of A-F adducts (additional ether
linkage type-A) was identified by nuclear magnetic resonance
(NMR) and their presence was confirmed in red wines.[11,17,22]
This work aimed to study the reactivity of the malvidin-3-
glucoside-(O)-catechin adduct (previously synthesized) with
catechin or mv3glc, both acetaldehyde-mediated, in order to
simulate putative reactions that take place in red wines.
Direct reactions between anthocyanins and flavan-3-ols
originate the dimeric-type flavanol-(4,8)-anthocyanin (F-A) and
anthocyanin-(4,8)-flavanol (A-F) adducts. The characterization
EXPERIMENTAL
Reagents
* Correspondence to: Victor de Freitas, Centro de Investigação
em Química, Departamento de Química e Bioquímica,
Faculdade de Ciências, Universidade do Porto, Rua do
Campo Alegre, 687, 4169-007 Porto, Portugal.
E-mail: vfreitas@fc.up.pt
(+)-Catechin was purchased from Sigma-AldrichW (Madrid,
Spain). Malvidin-3-glucoside (mv3glc) was isolated from a
young red table wine (Vitis vinifera L. cv. Touriga Nacional)
by semi-preparative HPLC using a reversed-phase C18
Rapid Commun. Mass Spectrom. 2012, 26, 2123–2130
Copyright © 2012 John Wiley & Sons, Ltd.

L. Cruz, N. Mateus and V. de Freitas
column (250 mm  4.6 mm i.d.) as reported elsewhere,[8]
and its structure and purity were confirmed by NMR.
TSK Toyopearl gel HW-40(S) was purchased from Tosoh
(Tokyo, Japan). Acetaldehyde was obtained from Fluka
Chemika (Buchs, Switzerland).
À18 ꢀC. The compound was further characterized by LC-
DAD/ESI-MS and its structure and purity assessed by NMR
analyses. The NMR data fits exactly with the that reported in
the literature.[17]
Hemisynthesis of malvidin-3-glucoside-(O)-catechin
Reactions of malvidin-3-glucoside-(O)-catechin with
catechin (or mv3glc) mediated by acetaldehyde
In order to maximize the yield of malvidin-3-glucoside-(O)-
catechin, several experimental conditions were studied,
namely pH, solvent, the temperature/time binomial and
molar ratio of malvidin-3-glucoside and (+)-catechin. A solu-
tion containing mv3glc (2.3 mM):(+)-catechin (molar ratio of
1:20) was prepared in water (200 mL) at pH 2.5 (adjusted with
dilute HCl or NaOH), protected from light and placed in the
oven at 50 ꢀC. The formation of the malvidin-3-glucoside-
(O)-catechin compound was monitored every day by high-
performance liquid chromatography (HPLC) with diode-array
detection (DAD) at 280 nm. After 7 days of incubation, the
synthetic reaction was stopped and the mixture purified.
Two solutions containing mv3glc-(O)-cat (1 mM):catechin
(or mv3glc):acetaldehyde (molar ratio of 1:4:10) were
prepared in 12% ethanol/water at pH 3.2 (adjusted with
dilute HCl or NaOH), protected from light and placed in
the oven at 30 ꢀC. The formation of the new compounds
was monitored by HPLC with DAD and by LC-DAD/ESI-MS
analysis in positive and negative ion mode.
HPLC
The samples were analyzed by HPLC (Merck-Hitachi L-7100)
on a 150 mm  4.6 mm i.d. reversed-phase C18 column
(Merck, Darmstadt, Germany) thermostatted at 25 ꢀC; detec-
tion was carried out at 280 nm using a diode-array detector
(Merck-Hitachi L-7450A). Solvents were (A) water/formic
acid 9:1 (v/v) and (B) acetonitrile, with the following
gradient: 10–35% B over 50 min at a flow rate of 0.5 mL/min.
The sample injection volume was 20 mL. The chromato-
graphic column was washed with 100% B during 10 min
and then stabilized with the initial conditions during another
10 min.
The semi-preparative HPLC system (Elite LaChrom) was
composed of a L-2130 quaternary pump, a manual injector
with loop of 1 mL and a L-2420 UV-vis detector. The station-
ary phase was composed of a reversed-phase C18 column
(Merck, Darmstadt, Germany) (250 mm  4.6 mm i.d., 5 mm
pore size) and the eluents were (A) water/formic acid 9:1
(v/v) and (B) acetonitrile, with the following gradient:
10–35% B over 50 min at a flow rate of 1 mL/min. Detection
wavelength was set to 280 nm.
Purification of malvidin-3-glucoside-(O)-catechin
The reaction mixture was extracted with ethyl acetate in order
to remove the catechins and other organic impurities. The
aqueous phase containing the desired compound was depos-
ited onto a medium-porosity sintered glass funnel loaded with
silica-C18 (reversed phase) connected to standard vacuum
filtration glassware. The solution was washed with water and
then eluted with methanol acidified with 2% HCl, yielding a
more concentrated volume. Next, semi-preparative HPLC was
performed in order to isolate the malvidin-3-glucoside-(O)-
catechin compound. The final purification was made by
column chromatography using TSK Toyopearl HW-40 (S) gel
(250 mm  16 mm i.d.) connected to a UV detector coupled to
a computer. The flow rate was regulated at 0.8 mL/min using
a peristaltic pump. The elution was performed with 70%
aqueous acidified methanol and the detection wavelength
was set to 280 nm. After removal of methanol on a rotary eva-
porator, the compound was freeze-dried and stored at
Figure 1. PDA chromatogram obtained from the LC-DAD analysis of the
aqueous fraction obtained from the reaction between malvidin-3-glucoside
and catechin. Inset: UV-vis spectrum of peak X (m/z 783).
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Rapid Commun. Mass Spectrom. 2012, 26, 2123–2130

LC-DAD/ESI-MS analysis of new methylmethine-linked adducts
The mass detector was a Finnigan LCQ DECA XP MAX
(Finnigan Corp., San Jose, CA, USA) quadrupole ion trap
equipped with an atmospheric pressure ionization (API)
source, using an electrospray ionization (ESI) interface. The
vaporizer and the capillary voltages were 5 kV and 4 V,
respectively. The capillary temperature was set at 325 ꢀC.
Nitrogen was used both as sheath and auxiliary gas at flow
rates of 90 and 25, respectively (in arbitrary units). Spectra
were recorded in positive and negative ion mode between
m/z 250 and 1500.
Table 1. Mass data of peak X (mv3glc-(O)-cat) obtained
from the ESI-MS analysis in the positive ion mode
Compound
[M + H]+ m/z
MS2 fragments
451 (À332)
469 (À314)
495 (À288)
621 (À162)
631 (À152)
657 (À126)
747 (À36)
mv3glc-(O)-cat
783
RESULTS AND DISCUSSION
LC-DAD/ESI-MS analysis
Hemisynthesis of malvidin-3-glucoside-(O)-catechin
A Finnigan Surveyor series liquid chromatograph, equipped
with a Thermo Finnigan (Hypersil Gold) reversed-phase
column (150 mm  4.6 mm, 5 mm, C18) thermostatted at
25 ꢀC, was used. The samples were analyzed using the same
solvents, gradients, injection volume and flow rate referred
above for HPLC analysis. Double-online detection was done
by a photodiode spectrophotometer and mass spectrometry.
The reaction of malvidin-3-glucoside (mv3glc) with (+)-catechin
(molar ratio 1:20) was performed in order to synthesize the
mv3glc-catechin dimeric structure. The reaction was followed
by HLPC and after 7 days of incubation the reaction was
stopped. After the work-up and the purification procedures
the sample was analyzed by LC-DAD/MS in the positive ion
mode (Fig. 1).
OMe
OMe
OMe
OH
OH
OH
+
HO
O
HO
O
OMe
OMe
OH
HO
O
2
OMe
OH
Oglc
+
δ
Oglc
O
4
OH
OH
Oglc
O
OH
OH
OH
OH
HO
8
OH
O
7
-
δ
HO
O
OH
OH
-
OH
δ
OH
OH
[M+H]+ m/z 783
OH
Figure 2. Formation pathway of the mv3glc-(O)-cat adduct (m/z 783).
Figure 3. PDA chromatogram obtained from the LC-DAD analysis in the negative
ion mode of the reaction of mv3glc-(O)-cat, (+)-catechin and acetaldehyde (1:4:10).
Inset: Full LC/MS spectra of peaks A–D (m/z 1097).
Rapid Commun. Mass Spectrom. 2012, 26, 2123–2130
Copyright © 2012 John Wiley & Sons, Ltd.
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L. Cruz, N. Mateus and V. de Freitas
The PDA chromatogram showed that the chromatographic
peak identified as peak X presents a pseudo-molecular ion
([M + H]+) at m/z 783 and MS2 fragments that agree with the
structure of the compound constituted by a mv3glc moiety
linked to a catechin molecule (Table 1).
The UV-vis spectrum of peak X (lmax 274 nm) revealed that
it is a colorless compound. After the compound isolation by
semi-preparative HPLC, the full 1 H and 13 C NMR characteri-
zation confirmed that it corresponds to a mv3glc-(4,8)-catechin
dimeric structure in its bicyclic form (additional ether linkage
type-A, C2-O-C7), which agrees with the data already
published for the mv3glc-(O)-(À)-epicatechin compound.[17]
This kind of structure suggests that the formation pathway of
this dimeric compound preferentially evolves towards to the
formation of the bicyclic form (more stable structure) rather
than to the cationic flavylium form (Fig. 2).
The major MS2 fragments obtained were as follows: m/z 631
resulting from retro-Diels-Alder (RDA) fission of the catechin
unit ([M + H–152]+), m/z 621 resulting from the loss of a
glucose residue ([M + H–162]+) and m/z 657 corresponding
to the loss of 126 u ([M + H–126]+) and its aglycone, m/z 495
([M + H–126–162]+).
OH
HO
m/z 1097
8
OMe
O
OH
OH
HC CH3
HO
HO
O
OH
OMe
OH
8
Oglc
OH
OH
O
O
-316 (cat-Mm)
-290 (cat)
OH
OH
OMe
OH
OMe
OH
CH2
O
HC
HO
O
OMe
OH
HO
OMe
OH
Oglc
OH
OH
O
Oglc
OH
OH
O
O
O
m/z 781
OH
m/z 807
OH
OH
OH
Figure 4. Fragmentation pattern of the cat-(8,8)-Mm-mv3glc-(O)-cat adduct in the negative ion mode.
Figure 5. PDA chromatogram obtained from the LC-DAD analysis of the reaction
mv3glc-(O)-cat, mv3glc and acetaldehyde (1:4:10). Inset: Full LC/MS spectra of
peaks G–L (m/z 1301).
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LC-DAD/ESI-MS analysis of new methylmethine-linked adducts
OH
m/z 1149
glc
O
OH
m/z 493
glc
O
MeO
HO
OMe
O
+
OH
MeO
HO
OH
O
+
HC CH3
O
OH
HO
OMe
OMe
OMe
-808 (Mm-mv3glc-(O)-cat)
O
glc
OH
O
O
OH
glc
O
-152 (RDA)
CH2
MeO
HO
8
OH
OMe
O
+
OH
OH
HC CH3
HO
O
OMe
OMe
OH
8
O
O
glc
OH
OH
O
m/z 1301
OH
OH
-782 (mv3glc-(O)-cat)
-162 (glucose)
OH
OH
glc
O
HO
MeO
HO
O
+
OH
MeO
HO
OMe
O
+
OH
m/z 519
HC
OH
HC CH3
O
CH2
OMe
HO
OMe
OMe
OH
-162 (glucose)
Oglc
O
OH
OH
O
OH
m/z 1139
HO
OH
MeO
HO
O
+
OH
OH
m/z 357
HC
CH2
OMe
Figure 6. Fragmentation pattern of the mv3glc-(8,8)-Mm-mv3glc-(O)-cat adduct in the positive ion mode.
Hemisynthesis of malvidin-3-glucoside-(O)-catechin-derived
compounds
leading to the formation of the adduct linked by the methyl-
methine bridge. This type of compounds contribute to the evo-
lution of red wine color during ageing and storage and that is
why their occurrence and formation have been widely studied.
In this work, reactions of malvidin-3-glucoside-(O)-catechin
with catechin and with malvidin-3-glucoside mediated by
acetaldehyde were separately performed in order to investigate
the formation of compounds with methylmethine bridges. It is
expected that each reaction will occur by the formation of
several positional isomers at C-6 or C-8 of the upper unit
methylmethine linked to two C-6 positions or one C-8 position
of the lower unit. Furthermore, two diastereoisomers may be
obtained for each positional isomer differing in the config-
uration of the asymmetric carbon of the methylmethine
bridge (R/S).[6,28,29]
It is well known that in red wines or in model solutions, flavan-
3-ols and anthocyanins, in the presence of acetaldehyde,
condense with each other through methylmethine (Mm)
bridges.[23–26] According to the mechanism reported in the
literature,[7] in acidic medium acetaldehyde originates the
respective carbocation which readily undergoes nucleophilic
attacks from the C-6/C-8 (preferentially) catechin positions that
have a high negative charge density due to the presence of
hydroxyl groups in ortho and para positions.[27] Then a dehydra-
tion occurs giving rise to the formation of an intermediate
carbocation which then undergoes nucleophilic attacks from
the C-6/C-8 (preferentially) of the mv3glc (hydrated form)
Rapid Commun. Mass Spectrom. 2012, 26, 2123–2130
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L. Cruz, N. Mateus and V. de Freitas
OMe
OMe
OH
O
H
OH
H
HO
CH3
OH
H+
+
C
H3C
C
8
H3C
HO
O
HO
O
OMe
OH
OMe
OH
8
6
-H+
Oglc
Oglc
O
OH
OH
O
OH
OH
O
O
6
OH
OH
OH
OH
OH
OH
8
OH
6
glcO
HO
6
O
HO
8
MeO
HO
O
OH
+H+ / -H2O
OH
OH
OMe
OMe
H
CH3
OH
+
HO
O
8
OMe
OH
Oglc
OH
OH
O
O
OH
OH
8
OH
glcO
-H+
-H2O
MeO
OH
OMe
O
+
OH
(R/S)
HO
OH
*
C CH3
H
HO
8
HO
O
OMe
OMe
O
OH
(R/S)
OMe
OH
8
OH
*
H
C
CH3
O
HO
Oglc
O
[M]+ m/z 1301
OH
HO
OH
O
OH
OMe
OH
8
Oglc
OH
[M-H]- m/z 1097
OH
O
OH
O
OH
OH
OH
Figure 7. Proposed reaction mechanisms between mv3glc-(O)-cat and catechin, and between mv3glc-(O)-cat and
mv3glc, both mediated by acetaldehyde.
Reaction between malvidin-3-glucoside-(O)-catechin and
catechin mediated by acetaldehyde
shown by their UV-vis spectra (lmax 277 nm). The two most
common signals in the fragmentation pattern of the
pseudo-molecular ion were m/z 807 ([M–H–290]–) and 781
([M–H–316]–) which correspond to the loss of a catechin moiety
and a methylmethine-catechin unit, respectively (Fig. 4).
The evolution of the reaction between mv3glc-(O)-cat and
catechin mediated by acetaldehyde was followed by HLPC.
LC-DAD/MS analysis of the reaction mixture in the negative
ion mode was performed and six chromatographic peaks
(peaks A–D, Fig. 3) with the same pseudo-molecular ion
([M–H]– m/z 1097) were detected.
Reaction between malvidin-3-glucoside-(O)-catechin and
mv3glc mediated by acetaldehyde
The six chromatographic peaks detected with m/z 1097
should correspond to the six positional isomers that are
expected to be formed. The pseudo-molecular ions and the
respective MS2 fragmentations patterns are consistent with
the structure composed by a catechin molecule and a
mv3glc-(O)-catechin adduct linked by a methylmethine
bridge. The methymethine compounds formed are colorless
The reaction between mv3glc-(O)-cat and mv3glc mediated
by acetaldehyde was analyzed by LC-DAD/MS and the
chromatogram revealed six peaks (peaks G–L, Fig. 5) with
the same molecular ion ([M]+ m/z 1301).
The UV-vis spectra of these peaks revealed that the new
compounds formed have a purple-red color (lmax 544 nm)
and their mass data is consistent with a compound composed
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Rapid Commun. Mass Spectrom. 2012, 26, 2123–2130

LC-DAD/ESI-MS analysis of new methylmethine-linked adducts
[3] J. He, C. Santos-Buelga, A. M. S. Silva, N. Mateus, V. De
Freitas. Isolation and structural characterization of new
anthocyanin-derived yellow pigments in aged red wines.
J. Agric. Food. Chem. 2006, 54, 9598.
[4] C. Dallas, J. M. Ricardo-Da-Silva, O. Laureano. Products
formed in model wine solutions involving anthocyanins,
procyanidin B-2, and acetaldehyde. J. Agric. Food. Chem.
1996, 44, 2402.
[5] N. E. Es-Safi, H. Fulcrand, V. Cheynier, M. Moutounet.
Studies on the acetaldehyde-induced condensation of
(À)-epicatechin and malvidin 3-O-glucoside in a model
solution system. J. Agric. Food Chem. 1999, 47, 2096.
[6] J. C. Rivas-Gonzalo, S. Bravo-Haro, C. Santos-Buelga.
Detection of compounds formed through the reaction of
malvidin-3-monoglucoside and catechin in the presence of
acetaldehyde. J. Agric. Food Chem. 1995, 43, 1444.
[7] C. F. Timberlake, P. Bridle. Interactions between anthocyanins,
phenolic compounds and acetaldehyde and their significance
in red wines. Am. J. Enol. Vitic. 1976, 27, 97.
[8] J. Pissarra, N. Mateus, J. C. Rivas-Gonzalo, C. Santos-Buelga,
V. De Freitas. Reaction between malvidin 3-glucoside and
(+)-catechin in model solutions containing different
aldehydes. J. Food Sci. 2003, 68, 476.
[9] Y. Hayasaka, J. A. Kennedy. Mass spectrometric evidence
for the formation of pigmented polymers in red wine. Aust.
J. Grape Wine Res. 2003, 9, 210.
[10] L. Jurd. Review of polyphenol condensation reactions and
their possible ocurrence in aging of wines. Am. J. Enol. Vitic.
1969, 20, 195.
[11] S. Remy, H. Fulcrand, B. Labarbe, V. Cheynier, M. Moutou-
net. First confirmation in red wine of products resulting
from direct anthocyanin-tannin reactions. J. Sci. Food Agric.
2000, 80, 745.
of the mv3glc (flavylium form) molecule and the mv3glc-(O)-
cat adduct linked by a methylmethine bridge. Some molecu-
lar fragments released are in agreement with the proposed
structure, namely m/z 1149 ([M–152]+), 1139 ([M–162]+), 519
([M–782]+), and 493 ([M–808]+), which correspond to RDA
fission, loss of a glucose residue, loss of the mv3glc-(O)-catechin
adduct and loss of a methylmethine-mv3glc-(O)-cat moiety,
respectively (Fig. 6).
Formation mechanism
According to the mechanism described in the literature,[7] in
acidic medium acetaldehyde attacks the phloroglucinol rings
of the mv3glc-(O)-cat which presents a negative charge
density in carbons C-6/C-8 (preferentially), to give the
respective ethanol-mv3glc-(O)-cat adduct. Further dehydra-
tion gives rise to the formation of an intermediate carbocation
which then undergoes nucleophilic attacks from the C-6/C-8
of the mv3glc in hydrated form or from the C-6/C-8 of the
catechin leading to the formation of the respective adducts
linked by methylmethine bridges (Fig. 7).
The proposed structures with 8,8-methylmethine displayed
in Fig. 7 are probably the ones that preferentially formed
bridges because the C-8 carbon possesses a higher negative
charge density than the C-6 position[27,30] and their formation
should thus be favored. The formation of several compounds
in low yields did not allow their isolation to proceed to
complete the structural elucidation by NMR.
[12] T. C. Somers. The polymeric nature of wine pigments.
Phytochemistry 1971, 10, 2175.
CONCLUSIONS
[13] E. Haslam, T. H. Lilley. Natural astringency in foodstuffs – a
molecular interpretation. Crit. Rev. Food Sci. Nutr. 1988, 27, 1.
[14] H. L. Wildenradt, V. L. Singleton. The production of aldehydes
as a result of oxidation of polyphenolic compounds and its
relation to wine aging. Am. J. Enol. Vitic. 1974, 25, 119.
[15] M. Duenas, E. Salas, V. Cheynier, O. Dangles, H. Fulcrand.
UV-visible spectroscopic investigation of the 8,8-methyl-
methine catechin-malvidin 3-glucoside pigments in
aqueous solution: Structural transformations and molecular
complexation with chlorogenic acid. J. Agric. Food. Chem.
2006, 54, 189.
The studies performed in model solutions showed that the
colorless mv3glc-(O)-cat adduct can undergo some of the
characteristic reactions of anthocyanins and flavan-3-ols that
occur in red wines in the presence of acetaldehyde forming
methylmethine-bridged compounds. The structures of the
resulting compounds were confirmed by their mass fragmen-
tation patterns and a formation mechanism was proposed.
These results bring more insights on the putative complexity
of the polyphenolic composition of red wines and opens
new pathways to discover more complex and polymeric
compounds in such matrices.
[16] H. Liao, Y. Cai, E. Haslam. Polyphenols interactions. Antho-
cyanins: Copigmentation and colour changes in red wines.
J. Sci. Food Agric. 1992, 59, 299.
[17] S. Remy-Tanneau, C. Le Guerneve, E. Meudec, V. Cheynier.
Characterization of
a colorless anthocyanin-flavan-3-ol
Acknowledgements
dimer containing both carbon-carbon and ether interflava-
noid linkages by NMR and mass spectrometry. J. Agric.
Food. Chem. 2003, 51, 3592.
The authors thank Dra. Zélia Azevedo for the LC-DAD/ESI-MS
analysis. Luís Cruz gratefully acknowledges the Postdoctoral
Grant from FCT (SFRH/BPD/72652/2010).
[18] L. Jurd. Anthocyanidins and related compounds. XI. Catechin-
flavylium salt condensation reactions. Tetrahedron 1967, 23, 1057.
[19] M. Mirabel, C. Saucier, C. Guerra, Y. Glories. Copigmenta-
tion in model wine solutions: Occurrence and relation to
wine aging. Am. J. Enol. Vitic. 1999, 50, 211.
REFERENCES
[20] C. Santos-Buelga, S. Bravo-Haro, J. C. Rivas-Gonzalo. Inter-
actions between catechin and malvidin-3-monoglucoside in
model solutions. Z. Lebensm.-Unters.-Forsch. 1995, 269.
[21] C. Santos-Buelga, E. M. Francia-Aricha, S. De Pascual-Teresa,
J. C. Rivas-Gonzalo. Contribution to the identification
of the pigments responsible for the browning of
anthocyanin-flavanol solutions. Eur. Food Res. Technol.
1999, 209, 411.
[1] J. Bakker, C. F. Timberlake. Isolation, identification, and
characterization of new color-stable anthocyanins occurring
in some red wines. J. Agric. Food Chem. 1997, 45, 35.
[2] H. Fulcrand, C. Benabdeljalil, J. Rigaud, V. Cheynier,
M. Moutounet. A new class of wine pigments generated
by reaction between pyruvic acid and grape anthocyanins.
Phytochemistry 1998, 47, 1401.
Rapid Commun. Mass Spectrom. 2012, 26, 2123–2130
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

L. Cruz, N. Mateus and V. de Freitas
[22] P. D. Bishop, C. W. Nagel. Characterization of the condensa-
tion product of malvidin-3,5-diglucoside and catechin.
J. Agric. Food. Chem. 1984, 32, 1022.
[23] T. Escribano-Bailon, M. Alvarez-Garcia, J. C. Rivas-Gonzalo,
F. J. Heredia, C. Santos-Buelga. Color and stability of
pigments derived from the acetaldehyde-mediated conden-
sation between malvidin 3-O-glucoside and (+)-catechin.
J. Agric. Food. Chem. 2001, 49, 1213.
[24] N. E. Es-Safi, V. Cheynier, M. Moutounet. Role of aldehydic
derivatives in the condensation of phenolic compounds
with emphasis on the sensorial properties of fruit-derived
foods. J. Agric. Food. Chem. 2002, 50, 5571.
[25] N. E. Es-Safi, H. Fulcrand, V. Cheynier, M. Moutounet.
Competition between (+)-catechin and (À)-epicatechin in
acetaldehyde-induced polymerization of flavanols. J. Agric.
Food. Chem. 1999, 47, 2088.
[26] C. Saucier, D. Little, Y. Glories. First evidence of acetalde-
hyde-flavanol condensation products in red wine. Am. J.
Enol. Vitic. 1997, 48, 370.
[27] G. Bendz, O. Martensson, E. Nilsson. Studies of flavylium
compounds. I. Some flavylium compounds and their prop-
erties. Arkiv Kemi. 1967, 27, 67.
[28] T. Escribano-Bailón, O. Dangles, R. Brouillard. Coupling
reactions between flavylium ions and catechin. Phytochemistry
1996, 41, 1583.
[29] D. F. Lee, E. E. Swinny, G. P. Jones. NMR identification of
ethyl-linked anthocyanin-flavanol pigments formed in
model wine ferments. Tetrahedron Lett. 2004, 45, 1671.
[30] V. Atanasova, H. Fulcrand, C. Le Guerneve, W. Cheynier,
M. Moutounet. Structure of a new dimeric acetaldehyde
malvidin 3-glucoside condensation product. Tetrahedron
Lett. 2002, 43, 6151.
wileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2012, 26, 2123–2130