A comprehensive investigation of guaiacyl-pyranoanthocyanin synthesis by one-/two-dimensional NMR and UPLC-DAD-ESI-MSn

Article abstract of DOI:10.1016/j.foodchem.2015.12.089

In red and rosé wines, the grape anthocyanins are progressively converted to more stable pigments, including phenylpyranoanthocyanins. One-/two-dimensional NMR and UPLC-DAD-ESI-MSⁿ measurements were used to monitor the synthesis of guaiacylpyranomalvidin 3-O-glucoside from malvidin 3-O-glucoside and vinylguaiacol in model solutions and identify the products formed during the reaction. The highest conversion rates (30%, determined by ¹H qNMR) were obtained with a small excess of vinylguaiacol in methanol/water (70/30) at pH 3 and 35 °C. Two reaction pathways competed with the formation of guaiacylpyranomalvidin 3-O-glucoside. The first one only concerns malvidin 3-O-glucoside and consists in C-ring cleavage with formation of malvone and smaller molecular weight breakdown products. This pathway is favored at higher pH and incubation temperature. At lower pH values or in the presence of large vinylguaiacol excess, faster consumption of malvidin 3-O-glucoside resulted from the formation of more complex pyranoanthocyanins substituted by vinylguaiacol oligomers.

Full text of DOI:10.1016/j.foodchem.2015.12.089

Food Chemistry 199 (2016) 902–910
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
A comprehensive investigation of guaiacyl-pyranoanthocyanin synthesis
by one-/two-dimensional NMR and UPLC–DAD–ESI–MSn
a
a
a
Anna Vallverdú-Queralt a,b, , Emmanuelle Meudec , Nayla Ferreira-Lima , Nicolas Sommerer ,
⇑
Olivier Dangles c, Véronique Cheynier a, Christine Le Guernevé a
a INRA, UMR1083 Sciences pour l’œnologie, Plateforme Polyphénols, 2, place Viala, Montpellier Cedex 34060, France
b Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid 28029, Spain
c Université d’Avignon, UMR408 Sécurité & Qualité des Produits d’Origine Végétale, 84000 Avignon, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 October 2015
Received in revised form 23 December 2015
Accepted 24 December 2015
Available online 24 December 2015
In red and rosé wines, the grape anthocyanins are progressively converted to more stable pigments,
including phenylpyranoanthocyanins. One-/two-dimensional NMR and UPLC–DAD–ESI–MSn measure-
ments were used to monitor the synthesis of guaiacylpyranomalvidin 3-O-glucoside from malvidin 3-
O-glucoside and vinylguaiacol in model solutions and identify the products formed during the reaction.
The highest conversion rates (30%, determined by 1H qNMR) were obtained with a small excess of vinyl-
guaiacol in methanol/water (70/30) at pH 3 and 35 °C. Two reaction pathways competed with the forma-
tion of guaiacylpyranomalvidin 3-O-glucoside. The first one only concerns malvidin 3-O-glucoside and
consists in C-ring cleavage with formation of malvone and smaller molecular weight breakdown prod-
ucts. This pathway is favored at higher pH and incubation temperature. At lower pH values or in the pres-
ence of large vinylguaiacol excess, faster consumption of malvidin 3-O-glucoside resulted from the
formation of more complex pyranoanthocyanins substituted by vinylguaiacol oligomers.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
NMR
UPLC–DAD–ESI–MSn
Guaiacylpyranomalvidin 3-O-glucoside
Optimization
Synthesis
1. Introduction
required to ensure complete conversion into the flavylium form
and good separation of the anthocyanins (Vrhovsek et al., 2012),
Numerous quality traits of plants and their transformation
products are related to the presence of secondary metabolites. In
particular, the color of most red and blue plant organs and of foods
and beverages made from them, such as rosé and red wines, is due
to anthocyanins. Anthocyanins are usually represented as the red
flavylium cation and classically analyzed by its spectrophotometric
detection in the visible range (around 520 nm) after High Perfor-
mance Liquid Chromatography (HPLC) separation using acidified
solvents. More sensitive and selective methods using HPLC coupled
to triple quadrupole mass spectrometry (HPLC–QqQ-MS) and
detection of specific mass transitions in multiple reaction monitor-
ing (MRM) mode have been recently developed for quantification
of anthocyanin flavylium cations (Arapitsas, Perenzoni, Nicolini,
& Mattivi, 2012; Lambert et al., 2015). However, in mildly acidic
solutions like wine, other forms, namely the colorless hemiacetal
and yellow chalcones, resulting from reversible water addition to
flavylium cation, are predominant. Highly acidic eluents are
but they are poorly suited for HPLC–MS analysis of other phenolic
compounds because of signal suppression in the source of the mass
spectrometer (Lambert et al., 2015). Partial conversion of hydrated
forms to the flavylium cation under the conditions used for HPLC
analysis results in broadening of the HPLC peaks and detection of
additional mass signals corresponding to hydrated species.
During wine-making and aging, the native anthocyanins pro-
gressively disappear and yield new pigments, among which the
pyranoanthocyanins, that are resistant to hydration, are
a
particularly important class (De Freitas & Mateus, 2011). The first
pyranoanthocyanins to be discovered were phenylpyranoantho-
cyanins (Cameira-dos-Santos, Brillouet, Cheynier, & Moutounet,
1996) that were identified after hemisynthesis from malvidin-3-
O-glucoside (Mv3G) and vinylphenol (Fulcrand, Cameira dos
Santos, Sarni-Manchado, Cheynier, & Favre-Bonvin, 1996). A mech-
anism involving a cycloaddition process of the vinylphenol ethyle-
nic bond and both the C-4 and the hydroxyl at C-5 of the
anthocyanin, followed by oxidation and aromatization to form an
additional pyrane ring, was proposed (Fulcrand, Benabdeljalil,
Rigaud, Chenyier, & Moutounet, 1998). Other phenylpyranoantho-
cyanin derivatives, including 4-vinylphenol, 4-vinylguaiacol,
⇑
Corresponding author at: UMR1083 Sciences pour l’œnologie, Plateforme
Polyphénols, 2, place Viala, Montpellier Cedex 34060, France.
E-mail addresses: avallverdu@ub.edu, vallverd@supagro.inra.fr (A. Vallverdú-
Queralt).
http://dx.doi.org/10.1016/j.foodchem.2015.12.089
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

A. Vallverdú-Queralt et al. / Food Chemistry 199 (2016) 902–910
903
4-vinylcatechol and 4-vinylsyringol adducts of several antho-
cyanins, were also identified in wine (Hayasaka & Asenstorfer,
2002) and in other plant extracts (Lu, Foo, & Wong, 2002). Because
of their particular chromatic and physico-chemical properties and
potential role in wine color, phenylpyranoanthocyanins have been
much investigated. Their quantification in wine has been achieved
by HPLC coupled to diode array detection (DAD) (Wirth et al.,
2012) or QqQ-MS in the MRM mode (Arapitsas et al., 2012;
Lambert et al., 2015), using Mv3G, the major grape anthocyanin,
as the calibration standard. Phenylpyranoanthocyanins have been
synthesized in model solution from either vinylphenols (Fulcrand
et al., 1996; Hakansson, Pardon, Hayasaka, de Sa, & Herderich,
(VG) as a case study. In this paper, we compare UPLC–DAD–ESI–
MSn and NMR analysis for monitoring the synthesis of Gpyra-
noMv3G and propose qNMR as a universal method to determine
the different forms of Mv3G (the major anthocyanin in grapes
and wines) and its reaction products. We also use a comprehensive
strategy combining ultra-high performance liquid chromatography
coupled to diode array detection and electrospray ionization ion
trap mass spectrometry (UPLC–DAD–ESI–IT-MS) and two dimen-
sion NMR (i.e. homonuclear 1H diffusion ordered spectroscopy
(DOSY) and 1H–13C Heteronuclear Single Quantum Correlation
(HSQC) and Heteronuclear Multiple Bond Correlation (HMBC) anal-
ysis), for separation and identification of reaction products.
As far as we know, this is the first study to provide an accurate
quantification of the pyranoanthocyanins using qNMR as well as
full characterization of the reaction medium by a combination of
LC–DAD–MS and NMR. Tentative identification of new compounds
and information on the reaction pathways are also provided.
2003; Sarni-Manchado, Fulcrand, Souquet, Cheynier,
&
Moutounet, 1996; Schwarz & Winterhalter, 2003) or hydroxycin-
namic acids (Blanco-Vega, López-Bellido, Alía-Robledo, & Her
mosín-Gutiérrez, 2011; Quijada-Morín, Dangles, Rivas-Gonzalo, &
Escribano-Bailón, 2010; Schwarz, Wabnitz, & Winterhalter, 2003).
High conversion rates were reported for the former reactions on
the basis of peak areas at 280 nm or 506–510 nm after HPLC sepa-
ration (Blanco-Vega et al., 2011; Hakansson, Pardon, Hayasaka, de
Sa, & Herderich, 2003; Schwarz & Winterhalter, 2003). However,
the low recoveries after purification suggest that the reaction
yields were in fact lower. This may be due to the lack of suitable
calibration standards and/or to anthocyanin degradation, as
described in other studies. Indeed, numerous degradation products
of Mv3G have been described and some of them formally identified
(Furtado, Figueiredo, Chaves das Neves, & Pina, 1993; Hrazdina,
1970; Hrazdina & Franzese, 1974; Lopes et al., 2007) but they have
not been quantified in the reaction media. Quantitative analysis of
the reaction using chromatographic methods would require cali-
bration using standard compounds, many of which are unstable
and/or not commercially available. Quantitative 1H Nuclear Mag-
netic Resonance (qHNMR), enabling quantitative determination
of molecules in complex solutions without the need for identical
pure reference standards (Pauli, Gödecke, Jaki, & Lankin, 2012;
Simmler, Napolitano, McAlpine, Chen, & Pauli, 2014), appears a
suitable alternative for accurate quantification of reagents and
products in complex reaction media, enabling in situ reaction mon-
itoring and determination of reaction yields (Do, Olivier, Salisbury,
& Wager, 2011).
2. Materials and methods
2.1. Chemicals and samples
2.1.1. Chemicals
All solvents were of HPLC quality and all chemicals of p.a. grade.
Methanol (CH3OH), acetonitrile (CH3CN), hydrochloric acid (HCl)
and formic acid (HCO2H) were purchased from VWR Prolabo (Fon-
tenay sous Bois. France). VG, 2,4,6-trihydroxybenzaldehyde and
syringic acids were purchased from Sigma (Saint-Louis, MO,
USA). Mv3G chloride was purchased from Extrasynthèse (Genay,
France). All deuterated solvents were purchased from Euriso-top,
France. Certified (traceCert) gallic acid and dimethylsulfone
(DMS) were obtained from Fluka Analytical, Switzerland. Purified
deionised water (MilliQ purification system, Millipore, France)
was used for the preparation of all solutions.
2.2. Sample preparation
For UPLC–DAD–MS analysis, model solutions (Mv3G 1–4 mM,
VG to Mv3G molar ratios (1:2, 1:1, 2:1) were prepared in different
CH3CN/H2O or CH3OH/H2O mixtures (from 100/0 to 20/80). For
NMR analysis, Mv3G (2–5 mg) was dissolved in the following sol-
vent mixtures: CD3CN/H2O, CD3CN/D2O, CD3OD/H2O or CD3OD/
D2O, 50/50 V/V. Stock solutions (ꢀ100 mM) of VG were prepared
in either CD3CN or CD3OD. The concentration of both solutions
was determined by qNMR. Aliquots of VG solutions were added
to Mv3G to reach the desired molar ratio of VG to Mv3G. An appro-
priate volume of CD3CN or CD3OD was then added to reach a 70/30
V/V ratio of organic solvent to H2O (or D2O). The pH of the mixture
was adjusted with concentrated HCl to the desired value and incu-
bations were carried out at different temperatures (from 25 to
45 °C). The solutions were analyzed by UPLC–DAD–MS and/or
NMR at regular intervals until completion of the reaction.
The aim of our work was to optimize the formation of
phenylpyranoanthocyanins in wine-like model solution model
solution, using formation of guaiacylpyranomalvidin 3-O-
glucoside (GpyranoMv3G) (Fig. 1) from Mv3G and vinylguaiacol
2.3. UHPLC–MS/MS analysis
Chromatographic runs were performed on a Waters Acquity
UPLC–DAD system (Waters, Milford, MA, USA), on an Acquity
BEH C18 column (150 mm length, 1 mm internal diameter,
1.7 lm particle size; Waters) at 35 °C following other procedures
(Lambert et al., 2015; Vallverdú-Queralt, Verbaere, Meudec,
Cheynier, & Sommerer, 2015). The mobile phase consisted of
H2O/HCO2H (99/1, V/V) (solvent A) and CH3OH/HCO2H (99/1, V/
V) (solvent B). The flow rate was 0.08 mL/min. The elution program
was as follows: isocratic with 2% B (1 min), 2–30% B (1–10 min),
isocratic with 30% B (10–12 min), 30–75% B (12–25 min), 75–90%
Fig. 1. Structure of GpyranoMv3G.

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B (25–30 min), and isocratic with 90% B (30–35 min). The injection
2.4. NMR analysis
volume was 0.5
650 nm.
lL. UV–visible spectra were recorded from 200 to
All experiments were performed on an Agilent 500 MHz DD2
NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA)
operating at 500.05 MHz for 1H and 125.75 MHz for 13C and
equipped with a 5 mm indirect detection Z-gradient probe. The
chemical shifts were reported to that of the internal organic sol-
vent (MeCN: at 1.94 ppm and 118.7 ppm for 1H and 13C respec-
tively, MeOH: at 3.31 ppm and 49.1 ppm for 1H and 13C
respectively). All data were collected with active sample tempera-
ture regulation.
ESI–MS/MS analyses were performed with a Bruker Daltonics
Amazon (Bruker, Darmstadt, Germany) mass spectrometer,
hyphenated to the UPLC–DAD system and equipped with an elec-
trospray source and an ion trap mass analyser. The spectrometer
was operated in the negative ion mode (capillary voltage, 4.5 kV;
end plate off set, À500 V; temperature, 200 °C; nebuliser gas,
10 psi and dry gas, 5 L/min) and in the positive ion mode (capillary
voltage, 2.5 kV; end plate off set, À500 V; temperature, 200 °C;
nebuliser gas, 10 psi and dry gas, 5 L/min). Collision energy for
fragmentation used for MS2 experiments was set at 1.
Assignments of both proton and carbon resonances, identifica-
tion, and structure characterization of products were performed
Fig. 2. 1D 1H-NMR monitoring of GpyranoMv3G synthesis in MeOH/H2O 70/30 V/V, pH 3, 35 °C. At t = 0, 3.9 mM Mv3G + 5 mM VG. At t = 72 h, 1.1 mM GpyranoMv3G. Some
typical signals are assigned (f = flavylium; a,b = hemiacetal epimers; e,z = (E)-chalcone and (Z)-chalcone forms, respectively, as in Fig. 2 supplementary). The signal areas used
for quantification are underlined. Following symbols are used: s Mv3G, ⁄ VG, d GpyranoMv3G, . intermediate and e final degradation products of Mv3G.

A. Vallverdú-Queralt et al. / Food Chemistry 199 (2016) 902–910
905
using both 1D and 2D NMR, homonuclear 1H (COSY (COrrelation
SpectroscopY), ROESY (Rotating frame Overhauser Effect Spectro-
scopY), DOSY) and heteronuclear 1H/13C (HSQC and HMBC) exper-
iments. Data were processed and analyzed using VNMRJ software.
Absolute concentration measurements were carried out by
qNMR using an external calibration method (Sterling, Crouch,
Russell, & Calderón, 2013). 20 mg of DMS and 20 mg of gallic acid
were accurately weighed and dissolved in a precise volume of D2O
(5 mL) and DMSO-d6 (5 mL) respectively. After probe tuning, a sin-
gle scan 1D 1H spectrum of DMS was collected using a 90° pulse.
The surface area of DMS signal was used for probe calibration using
VNMRJ absolute intensity qNMR utility software. The probe
Fig. 3. Refined mechanism for GpyranoMv3G synthesis.

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calibration was then verified using the gallic acid sample. The cal-
ibration probe was checked on a regular basis.
of GpyranoMv3G at 506 nm to the initial peak area of Mv3G at
520 nm was 85%, and thus slightly higher than that reported earlier
(Hakansson et al., 2003). Changes in the Mv3G concentration (from
1 to 4 mM) resulted in no significant difference and changes in the
molar ratios (1:2, 2:1) resulted in lower yields.
The absolute concentration of Mv3G (under its different forms),
VG, and products formed during the synthesis were determined
from 4 to 32 scans 1D 1H spectra using a 90° pulse and an interscan
delay equal to 20 s (>5T1). Well-separated signals were used for
the surface signal integration.
Monitoring of the reaction by qNMR enabled accurate determi-
nation of the concentrations of reagents and products. At pH 3,
Mv3G was present as a mixture of flavylium, hemiacetal (2R and
2S epimers) (Jordheim, Fossen, & Andersen, 2006), (Z)-chalcone
and (E)-chalcone forms (Brouillard & Delaporte, 1977) in equilib-
rium (see assignments in Fig. 1 supplementary). The best reaction
yield, obtained in CD3OD/H2O (70/30, V/V), at pH 3 and 35 °C, with
a small excess of VG, was about 30% (Fig. 2 supplementary) and
thus much lower than that estimated by UPLC–DAD. Part of the
discrepancy can be explained by inaccuracies in the initial Mv3G
concentration (e.g. due to the presence of water or salts in the
Mv3G sample) in the UPLC–DAD–MS experiments. Moreover, esti-
mation of Mv3G from its peak area in the visible range results in
underestimation as part of the molecule is eluted under colorless
hydrated forms (hemiacetal, chalcones) under our chromato-
graphic conditions (1% HCO2H). However, our results suggest that
the response factor of GpyranoMv3G is much higher than that of
Mv3G at the maximum absorption wavelength in the visible range
(i.e. 506 and 520 nm, respectively) and emphasize the need for
accurate determination of the purity and response factors of
reagents and products for proper determination of reaction yields.
An example of reaction monitoring by 1H qNMR is presented in
Fig. 2. Complete assignment of the 1H and 13C signals by ROESY,
HSQC, and HMBC experiments allowed attribution of major signals
detected to Mv3G, VG, and GpyranoMv3G, as expected, although
other compounds also accumulated during the incubation (see
below).
3. Results and discussion
3.1. Optimization of reaction conditions
Preliminary experiments were carried out to select the best
conditions for conversion of Mv3G to GpyranoMv3G, which was
identified on the basis of its UV–visible and MS spectra (kmax
506 nm, m/z 639 in the positive ion mode, fragment ion at m/z
477, corresponding to the loss of the glucosyl moiety). The highest
yields, determined by monitoring the peak area of GpyranoMv3G
at 506 nm, were obtained with mixtures of H2O/MeCN (30/70)
and H2O/MeOH (30/70) acidified to pH 3. The reaction took longer
to complete at temperatures below 25 °C and temperatures above
35 °C led to increased degradation of both Mv3G and Gpyra-
noMv3G. Above pH 3, the formation of GpyranoMv3G decreased
significantly (P < 0.05) as more hemiacetal is formed at the expense
of the flavylium ion.
Consumption of Mv3G was faster at lower pH values. However,
production of GpyranoMv3G was very low at pH 1. In the absence
of VG, the loss of Mv3G was only about 10% after 96 h at pH 1 and
40 °C (vs. 100% after 20 h in the presence of VG), indicating that VG
is involved in its consumption. Under these reaction conditions
(2 mM Mv3G, 2 mM VG, pH 3, 35 °C), the ratio of the peak area
Fig. 4. Expansions of DOSY spectrum recorded during synthesis of GpyranoMv3G (MeCN/H2O 30/70 V/V, pH 3, 35 °C), showing some typical signals of Mv3G (s),
GpyranoMv3G (d) and malvone (e).

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907
3.2. NMR identification of major reaction products
2002), which in aerobic conditions, can form reactive oxygen spe-
cies (FeII–O2 M FeIII–O–OÅ) for subsequent H-atom abstraction.
Intermediate III must be especially prone to cyclization by
intramolecular 1,6-Michael addition and thus yields intermediate
IV, which again is highly sensitive to autoxidation with concomi-
tant aromatization. Interestingly, intermediate IV must permit fast
proton – deuterium exchange at C4 in agreement with the experi-
mental observation. Moreover, this mechanism accounts for the
formation of H2O2 in the medium, in agreement with the accumu-
lation of malvone, which has been formed by Baeyer–Villiger rear-
rangement after the nucleophilic attack of H2O2 onto the C2 center
of the flavylium ion (Hrazdina, 1970) (Fig. 3 supplementary).
Finally, it is also interesting to note that the non-cyclized interme-
diate III can be detected by UPLC–DAD–MS (see below).
DOSY analysis permits to discriminate compounds as a function
of their diffusion coefficients. Thus, 1H signals of Mv3G, Gpyra-
noMv3G and other products present in the reaction medium could
be distinguished, as illustrated in Fig. 4. The major product show-
ing a diffusion coefficient lower than that of Mv3G was formally
identified for the first time as malvone on the basis of NMR data
(Fig. 5 and Table 2 supplementary). The NMR spectra showed the
Chemical shifts of GpyranoMv3G are given in Table 1 Supple-
mentary. When the reaction was carried out in CD3OD/D2O
(70/30), the GpyranoMv3G 1H spectrum lacked the H9 signal.
Addition of H2O to the reaction medium did not allow the recovery
of this signal, meaning that the proton was no longer exchangeable
in the pyranoanthocyanin. In contrast, some exchange has been
reported earlier in the case of carboxypyranoanthocyanins
(Jordheim, Fossen, Songstadt, & Andersen, 2007) and 10-acetyl-
pyranoanthocyanins (Gómez-Alonso, Blanco-Vega, Gómez, & Her
mosín-Gutiérrez, 2012). A refined version for the mechanism of
pyranoanthocyanin synthesis is shown in Fig. 3. The first step is
a nucleophilic attack of VG onto the C4 center of the Mv3G flavy-
lium ion (1,4-Michael addition). The benzylic carbocation interme-
diate thus formed (I) can undergo either cyclization (attack of
Mv3G C5-OH) or proton loss. The latter route is preferred as it
leads to intermediate II, which displays a very labile H4-atom
allowing rapid autoxidation with a concomitant large extension
of conjugation (intermediate III). Such autoxidation is expected
to be initiated by unidentified FeII traces (Welch, Davis, & Aust,
Fig. 5. (A) Malvone structure. (B) 2D HSQC spectrum recorded during GpyranoMv3G formation (MeCN/H2O 70/30 V/V pH 1, 35 °C) showing 1J-CH correlations within
malvone. (C) Expansion of 2D HMBC spectrum recorded during GpyranoMv3G formation (MeCN/H2O 70/30 V/V pH 1, 35 °C) showing 2J-CH and 3J-CH correlations within
malvone implying H2B and H6B. (D) Expansion of 2D HMBC spectrum recorded during GpyranoMv3G formation (MeCN/H2O 70/30 V/V pH 1, 35 °C) showing 2J-CH and 3J-CH
correlations implying the following malvone protons: H4, H6, H8, H1Glc, OCH3.

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A. Vallverdú-Queralt et al. / Food Chemistry 199 (2016) 902–910
presence of two aromatic rings similar to those of Mv3G: two meta
coupled doublets in the aromatic region (6.2 and 6.3 ppm,
J = 1.8 Hz) could be assigned to the H6 and H8 (A-ring), whereas
an aromatic downfield singlet at 7.4 ppm (integrating for 2 pro-
tons) and a methoxy singlet at 3.9 ppm (integrating for 6 protons)
were typical of the B-ring protons. An anomeric glucose proton in b
configuration also appeared at 5.35 ppm (J = 8.3 Hz). The 1H/13C 2D
spectra were particularly useful for full structural characterization.
The most apparent difference between the malvone and Mv3G
spectra was a methylene signal, clearly observed on the edited-
HSQC spectrum (3.55 ppm for 1H, 30 ppm for 13C) of malvone
(Fig. 5C), instead of the downfield C4 signal of Mv3G. The attribu-
tion of quaternary carbons of A- and B-rings was straightforward
from HMBC spectrum (Fig. 5C and D). Besides, two carbonyl car-
bons were observed: one at 166 ppm which correlated with H2’
and H6’, was attributed to C2 whereas the other at 172 ppm gave
long-range correlations with both the anomeric glucose and vinyl
protons and was thus attributed to C3.
and consumption rates increased with the temperature and at lower
pH values and it was no longer detected at the end of the reaction.
Additional series of pigments with maximum absorbance at
506 nm or 515 nm were observed in the positive ion mode in solu-
tions incubated at pH 1. The main products, detected at m/z 787
(kmax 506 nm) and m/z 789 (kmax 515 nm), correspond to addition
of one VG dimer to Mv3G, respectively in the cyclized (pyrane)
and open forms (Fig. 4 supplementary). Ions detected at m/z 939
(kmax 515 nm), m/z 937 (kmax 506 nm) and m/z 1089 (kmax
515 nm) can similarly be attributed to pyranoanthocyanins result-
ing from reaction with VG trimer and tetramer. All these pigments
were also found in solutions containing a twofold excess of VG
incubated at pH 3. They may correspond to the polymeric material
mentioned in an earlier study using vinylphenol in excess, for
which no structure has been proposed (Hakansson et al., 2003).
Indeed, as an electron-rich styrene, VG is prone to acid-catalyzed
oligomerization and VG dimers were actually reported earlier
(Arrieta-Baez et al. 2012). Formation of complex pyranoantho-
cyanins derived from these oligomers competes with the main
reaction and may be responsible for rapid loss of Mv3G and lower
reaction yield obtained at pH 1 or in the presence of large VG
excess.
3.3. Tentative identification of minor products by UPLC–DAD–MS
UPLC–DAD–MS analysis of the different reaction media showed
the presence of additional pigments (detected as flavylium cations
in the positive ion mode) and of numerous other phenolic com-
pounds absorbing only in the UV range and detected in the nega-
tive ion mode.
A pigment (kmax 515 nm), detected at m/z 641 in the positive ion
mode after a few hours of incubation, was attributed to intermediate
III (Fig. 3), described earlier by Hakansson et al. (2003). Its formation
The UPLC profiles at 280 nm were much more complex. The
profile obtained at the end of the reaction at pH 3 and 35 °C is
shown in Fig. 6. Ions detected in the negative ion mode at m/z
509 and providing a fragment ion at m/z 347 (loss of a hexose moi-
ety) correspond to hydrated forms of Mv3G. Compound (A) dis-
played a maximum UV–vis absorption at 330 nm and was thus
attributed to one of the chalcone forms. The other mass signals
Fig. 6. Degradation products of Mv3G detected by LC–MS, at 280 nm at the end of the reaction (72 h, MeCN/H2O 70/30 V/V pH 1, 35 °C).

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909
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could be attributed to degradation products of Mv3G as summa-
rized in Fig. 6. Thus, a product detected at m/z 525 (compound B)
with a maximum UV–vis absorption at 283 nm and showing frag-
ment ions at m/z 345, and m/z 165, was assigned to malvone.
Another ion detected at m/z 363 (compound C) and showing the
same UV–visible spectrum and fragmentation pattern was attribu-
ted to malvone aglycone. Peaks detected at m/z 153 and at m/z 197
(compounds D and E), with a fragment ion at m/z 153, were
assigned to 2,4,6-trihydroxybenzaldehyde and syringic acid, which
are other known degradation products of malvidin derivatives aris-
ing from cleavage of the chalcone aglycone (Furtado et al., 1993) or
the malvone intermediate (Hrazdina & Franzese, 1974). Their iden-
tification was confirmed by co-injection of the corresponding stan-
dards. Signals detected at m/z at 345 (compound F), with fragment
ions at m/z 183 (loss of hexose) and m/z 165 (loss of water), and at
m/z 183 (compound G), with a fragment ion at 139 (loss of car-
boxylic group), can be tentatively attributed to 3-hydroxyphenyl
acetyl glucoside and 3-hydroxyphenylacetic acid. Two peaks
detected at at m/z 343 (compounds H and I) with fragment ions
at m/z 181 (loss of hexose) were attributed to cis- and trans-antho-
cyanone
A
(8-b-D-glucopyranosyl-2,4-dihydroxy-6-oxo-cyclo
hexa-2,4-dienylacetic acid) identified as other breakdown products
of malvone (Lopes et al., 2007). Another molecular ion at m/z 333
(compound K) gave a fragment at m/z 179, which probably results
from the loss of a 2,4,6-trihydroxybenzaldehyde moiety.
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In conclusion, the reaction conditions for the formation of Gpyr-
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tions. The highest conversion rate, accurately measured by
qNMR, was 30%. Two reaction pathways competed with the forma-
tion of GpyranoMv3G. Degradation of Mv3G via chalcone or mal-
vone intermediates led to smaller molecular weight breakdown
products. At lower pH or with VG in large excess, a faster consump-
tion of Mv3G was observed, due to formation of more complex
pyranoanthocyanins derived from VG oligomers. Our approach,
using UPLC–DAD–MS and NMR performed directly on the reaction
medium for monitoring the reaction, combined the sensitivity and
selectivity of MS analysis, which detects molecules present in
small concentration, and the accuracy of NMR, which enables
quantification and formal identification of the major products.
Hakansson, A. E., Pardon, K., Hayasaka, Y., de Sa, M., & Herderich, M. (2003).
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Hayasaka, Y.,
& Asenstorfer, R. E. (2002). Screening for potential pigments
derived from anthocyanins in red wine using nanoelectrospray tandem mass
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under acidic and neutral conditions. Phytochemistry, 13, 231–234.
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&
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A high-throughput UHPLC-QqQ-MS method for
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Anthocyanone A: A quinone methide derivative resulting from malvidin 3-O-
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2698–2704.
Acknowledgments
We thank Jean Paul Mazauric for the technical support. Anna
Vallverdú-Queralt is grateful to the Alfonso Martín Escudero Foun-
dation for the postdoctoral fellowship for carrying out research
abroad. Funding for this work was provided by the Instituto de
Salud Carlos III, ISCIII (CIBEROBN), the Quality Group from Gener-
alitat de Catalunya (GC) 2014 SGR 773 and by INRA Departement
CEPIA. Financial support from GIS IBiSA (Infrastructures en Biologie
Santé et Agronomie), Région Languedoc Roussillon, and INRA CNOC
for funding of the UPLC–MS and NMR equipment is also
acknowledged.
Lu, Y., Foo, L. Y., & Wong, H. (2002). Isolation of the first C-2 addition products of
anthocyanins. Tetrahedron Letters, 43, 6621–6624.
Pauli, G. F., Gödecke, T., Jaki, B. U., & Lankin, D. C. (2012). Quantitative 1H NMR:
Development and potential of an analytical method – An update. Journal of
Natural Products, 75, 834–851.
Quijada-Morín, N., Dangles, O., Rivas-Gonzalo, J. C., & Escribano-Bailón, M. T. (2010).
Physico-chemical and chromatic characterization of malvidin 3-glucoside-
vinylcatechol and malvidin 3-glucoside-vinylguaiacol wine pigments. Journal of
Agricultural and Food Chemistry, 58, 9744–9752.
Sarni-Manchado, P., Fulcrand, H., Souquet, J.-M., Cheynier, V., & Moutounet, M.
(1996). Stability and color of unreported wine anthocyanin-derived pigments.
Journal of Food Science, 61, 938–941.
Schwarz, M., & Winterhalter, P. (2003). A novel synthetic route to substituted
pyranoanthocyanins with unique colour properties. Tetrahedron Letters, 44,
7583–7587.
Appendix A. Supplementary data
Schwarz, M., Wabnitz, T. C., & Winterhalter, P. (2003). Pathway leading to the
formation of anthocyanin-vinylphenol adducts and related pigments in red
wines. Journal of Agricultural and Food Chemistry, 51, 3682–3687.
Simmler, C., Napolitano, J. G., McAlpine, J. B., Chen, S. N., & Pauli, G. F (2014).
Universal quantitative NMR analysis of complex natural samples. Current
Opinion in Biotechnology, 25, 51–59.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
12.089.
Sterling, C., Crouch, R., Russell, D. J., & Calderón, A. I. (2013). 1H-NMR quantification
of major saccharides in Açaí Raw materials: A comparison of the internal
standard methodology with the absolute intensity qNMR method.
Phytochemical Analysis, 24, 631–637.
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