Identification of autoxidation oligomers of flavan-3-ols in model solutions by HPLC-MS/MS

Article abstract of DOI:10.1002/jms.1536

Autoxidation of flavan-3-ols was carried out in aqueous/methanol model solutions under mildly acidic conditions (pH 6.0), and these autoxidation products were analyzed by using high performance liquid chromatography (HPLC) coupled with tandem mass spectro

Full text of DOI:10.1002/jms.1536

Research Article
Received: 4 July 2008
Accepted: 28 October 2008
Published online in Wiley Interscience: 3 December 2008
(www.interscience.com) DOI 10.1002/jms.1536
Identification of autoxidation oligomers of
flavan-3-ols in model solutions by HPLC-MS/MS
Fei He,† Qiu-Hong Pan,† Ying Shi, Xue-Ting Zhang and Chang-Qing Duan∗
Autoxidation of flavan-3-ols was carried out in aqueous/methanol model solutions under mildly acidic conditions (pH 6.0), and
these autoxidation products were analyzed by using high performance liquid chromatography (HPLC) coupled with tandem
mass spectrometry (MS/MS). The results showed that (+)-catechins and (−)-epicatechins generated autoxidation reaction with
each other to form a series of oligomers that had the same [M − H]− molecular ions (MS1) as those of natural procyanidins,
but had completely different fragment ions (MS2). According to MS/MS analysis, the major fragments of these oligomers were
derived not only from the retro-Diels–Alder (RDA) dissociations on the C-rings of the flavan-3-ol units, but also from the
quinone-methide (QM) cleavage of the interflavan linkages (IFL), and thus they were identified as B-type dehydrodicatechins,
B-type dehydrotricatechins and A-type dehydrotricatechins, respectively. The potential structures of their [M − H]− molecular
ions and partial fragment ions were deduced on the basis of the MS/MS characterization and the oxidation of flavan-3-ols in
previous reports. Some specific fragment ions were found to be very useful for identifying the autoxidation oligomers (the
B-type dehydrodicatechins at m/z 393, the B-type dehydrotricatechins at m/z 681 and the A-type dehydrotricatechins at m/z
c
725). Copyright ꢀ 2008 John Wiley & Sons, Ltd.
Keywords: HPLC-DAD-ESI/MS; flavan-3-ols; autoxidation; B-type dehydrodicatechin; B-type dehydrotricatechin; A-type dehydrotricate-
chin
Introduction
the yellow compounds at pH > 4.0. In the autoxidation, similar
polymerization occurs very fast in alkaline medium and sometimes
takes place under acid conditions, especially when metal catalysts
are present.[6,9]
Flavan-3-ols are a class of phenolic secondary metabolites
synthesized via the flavonoid biosynthetic pathway in various
plants.[1] Flavan-3-ols exist widely in the plant kingdom and in
the plant-derived products, particularly in wines, where they
contribute not only to food organoleptic properties of the
products such as astringency, bitterness and color alternation,[2]
but also to food functionality including microbial stability,
foamability and oxidative stability.[3] Meanwhile, flavan-3-ols
also exhibit several health beneficial effects by acting as
antioxidant, anticarcinogen, antimicrobial, anti-inflammatory,
cardio-preventive, UV-protective and neuron-protective agents.[4]
As a result of enzymatic oxidation, chemical oxidation or
autoxidation of flavan-3-ols, a group of unnatural oligomers
are synthesized, which have the same molecular ions mass
spectrum (MS1) as their isomeric proanthocyanidins in natural
resources, but have completely different structures. Generally,
the oxidation dimers of flavan-3-ols linked by C6ꢁ → C8 or
C6ꢁ → C6 interflavan linkages (IFL) are classified as B-type
dehydrodicatechins, resulting from the repeated condensation
reactions between the A-ring of the lower unit and the B-ring of
the upper unit through a mechanism of so-called ‘head to tail’
polymerization.[5–7] And correspondingly, the oxidation dimers
which contain additional C–O–C ether-type IFL are classified as
A-type dehydrodicatechins.[8] The detailed structures of flavan-
3-ols and the oxidation dehydrodicatechins are illustrated in
Scheme 1. B-type dehydrodicatechins are colorless whereas A-
type dehydrodicatechins are yellow, and most of these dimers
could be formed by chemical oxidation of (+)-catechin under
the alkaline conditions. But in the polyphenol oxidase (PPO)-
mediated oxidation of (+)-catechin, the colorless condensed
products are formed in larger amounts at pH < 4.0 whereas
Although significant developments have been made to obtain
structural information on the natural oligomeric flavan-3-ols as
well as the oxidation dimers by using high pressure liquid
chromatography (HPLC) coupled with mass spectrometry (MS)
and/or nuclear magnetic resonance (NMR) during the past
decades, little is known about the synthesis and the structural
identification of the oxidation trimeric flavan-3-ols.[10–12] In the
present paper, we described the autoxidation of flavan-3-ols in
aqueous/methanolmodelsolutionsundermildlyacidicconditions
(pH 6.0). On the basis of the HPLC-diode array detection (DAD)-
ESI-MS/MS data reported previously, we identified a series of
B-type and A-type dimeric and trimeric autoxidation flavan-3-
ols and speculated their structures and dissociation mechanisms.
Furthermore, the specific fragment ions were chosen to develop a
rapid and sensitive method for distinguishing these autoxidation
oligomers from natural procyanidins.
∗
Correspondence to: Chang-Qing Duan, Centre for Viticulture and Enology,
College of Food Science and Nutritional Engineering, China Agricultural
University, Beijing 100083, China. E-mail: chqduan@yahoo.com.cn
†
These authors equally contributed to this work
Centre for Viticulture and Enology, College of Food Science and Nutritional
Engineering, China Agricultural University, Beijing 100083, China
c
J. Mass. Spectrom. 2009, 44, 633–640
Copyright ꢀ 2008 John Wiley & Sons, Ltd.

F. He et al.
Scheme 1. Detailed structures of flavan-3-ols [(+)-catechin and (−)-epicatechin) and oxidation dehydrodicatechins (B12 and A).
Experimental
(solvent B) applying the following gradient: 0–25 min: 10–22% B,
25–45 min: 22–50% B, 45–55 min: 50–95% B, 55–60 min: 95%
B isocratic, 60–63 min: 95–10% B, 63–66 min: 10% B isocratic.
The flow rate was 1.0 ml min−1. Injection volume was 10 µl with
the UV detector set to an absorbance wavelength of 280 nm.
The ESI parameters were as follows (optimized depending on
compounds): nebulizer, 30 psi; dry gas (N2) flow, 10 l min−1; and
dry gas temperature 325 ◦C; the ion trap mass spectrometer was
operated in negative ion mode with a scanning range from m/z
200 to 800. In addition, the activation energy for the MS/MS
experiment was set to 1.0 V.
Material
Methanolandglacialaceticacid(HPLCgrade)werepurchasedfrom
Fisher (Fairlawn, NJ, USA). Deionized water (<18 Mꢀ resistance)
was obtained from a Milli-Q Element water purification system
(Millipore, Bedford, MA). (+)-Catechin, (−)-epicatechin and all
the other chemicals were purchased from Sigma Chemical Co.
(St Louis, MI, USA) unless noted specially.
Preparation of autoxidation products of flavan-3-ols
(+)-Catechin and (−)-epicatechin were dissolved in methanol to
obtain their 5 and 10 mM methanol solutions, respectively. 2-(N-
Morpholino)ethanesulfonic acid (MES)-NaOH buffer (100 mM, pH
6.0) was prepared for further use as a reaction buffer.
Results and Discussion
Autoxidation of (+)-catechin
In the autoxidation of (+)-catechin, (+)-catechin methanol
solution (200 µl, 5 mM) was mixed with MES-NaOH buffer (800 µl,
100 mM) mentioned above to obtain the reaction system at pH
6.0. The final mixture (approximately 1 ml) was incubated in dark
at room temperature for 24 h, and then was filtered through
Gelman(AnnArbor, MI, USA)GHPolypro(GHP)Acrodisc13syringe
filters (0.45 µm) and analyzed through HPLC-DAD-ESI-MS/MS. The
autoxidation of (−)-epicatechin was conducted according to the
same method used for (+)-catechin. In the autoxidation mixture of
(+)-catechin and (−)-epicatechin, (+)-catechin methanol solution
(100 µl, 10 mM) was mixed with (−)-epicatechin methanol solution
(100 µl, 10 mM), and MES-NaOH buffer (800 µl, 100 mM) mentioned
above was then added to the mixture to obtain the reaction
systems at pH 6.0. The final mixtures (approximately 1 ml) were
incubated, filtered and analyzed adopting the same method
mentioned above.
In the autoxidation of (+)-catechin carried out in aque-
ous/methanol model solution under mildly acidic conditions (pH
6.0), besides the residual substrate (+)-catechin and the buffer
content MES, four new compounds were detected as shown in
Fig. 1(a). Among these products, peak 1 had the same [M − H]−
molecular ion at m/z 577 as the natural B-type procyanidin dimers,
but had the completely different fragment ions at m/z 533, 439,
425, 393 and 269. On the basis of the previous report of Sun
and Miller, we could easily identify this dimeric flavan-3-ols as
B-type dehydrodicatechin B11, which was condensed from two
(+)-catechin units.[14] However, there were still three unknown
products (peaks 2, 3, and 4), as shown in Table 1.
Autoxidation of (−)-epicatechin
Compared with the autoxidation of (+)-catechin, the autoxidation
of (−)-epicatechin yielded quite similar but more complex
results (Fig. 1(b)), as summarized in Table 1. Under the mildly
acidic conditions (pH 6.0), we obtained ten new compounds,
including (+)-catechin (peak 7) which was transformed from (−)-
epicatechin, and three unknown products (peaks 2, 3 and 4)
which were also present in the products of the autoxidation
of (+)-catechin, indicating that these products had lost their
chemical stereospecificity during the reactions and were formed
as the mutual oxidation products of stereoisomeric (+)-catechin
and (−)-epicatechin. Besides these, we obtained a series of
oxidation oligomers of flavan-3-ols, including three dimers and
three trimers.
HPLC-DAD-ESI-MS/MS analyses
Analysis of phenolic compounds was carried out according to the
method reported by Sun et al., by using an Agilent 1100 series LC
and LC/MSD Trap VL mass spectrometer (Agilent Technologies,
Palo Alto, CA, USA) equipped with electrospray ionization (ESI)
interface.[13] The LC system includes a G1379A on-line degasser,
a G1311A quaternary pump, an 1313A autosampler, a G1316A
thermostatic column control, and a G1315A DAD, all of which
were controlled by the Agilent ChemStation version 5.2 software.
The HPLC separation was performed on a reversed-phase Zorbax
SB-C18 column (250 mm × 4.6 mm i.d. 5-µm particle size, Agilent
Technologies, USA) at 25 ◦C. The mobile phase consisted of 1%
acetic acid in water (solvent A) and 1% acetic acid in methanol
All of the three dimeric flavan-3-ols, peaks 8, 9 and 10
had the typical [M − H]− molecular ions and fragment ions
c
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Copyright ꢀ 2008 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2009, 44, 633–640

MS/MS of autoxidation oligomers of flavan-3-ols
Figure 1. HPLC-ESI/MSn (base peak representation) traces of the phenolic products of the autoxidation of flavan-3-ols in aqueous/methanol model
solutions undermildly acidic conditions (pH 6.0):(a) the autoxidation of (+)-catechin;(b) the autoxidation of (−)-epicatechin;(c) the autoxidation mixtures
of (+)-catechin and (−)-epicatechin. Peak numbers correspond to the labeling adopted in Table 1.
of B-type dehydrodicatechin. This result was quite similar to
what Sun and Miller reported, and the latter obtained three
typical B-type dehydrodicatechin B11, B12 and B22 as well
as two unknown B-type dehydrodicatechins (both of them
were called By) from the 1 : 1 reaction of (+)-catechin and
(−)-epicatechin in 25 mM ammonium acetate solution (pH
6.5).[14] Generally, typical B-type dehydrodicatechin B11 and
B12 were thought to contain at least one (+)-catechin unit.
However, such typical B-type dehydrodicatechins were not
found in the present autoxidation of (−)-epicatechin. Therefore,
according to the characteristic of their retention times and
peak sequence which were similar to the previous report, we
primarily deduced that both peaks 8 and 9 might be the
unknown B-type dehydrodicatechin By and peak 10 might
be B-type dehydrodicatechin B22 which was condensed from
two (−)-epicatechin units. However, because the conversion
of (−)-epicatechin to (+)-catechin also occurred in the model
solution, we could not exclude the possibility that (+)-catechins
had also participated in the formation of these unknown
B-type dehydrodicatechins. Furthermore, we also speculated
that alternative C6ꢁ-C6 IFL might occur in these unknown
B-type dehydrodicatechins, which resulted in their different
configurations and retention times, but did not change their
[M−H]− molecularionsandfragmentions.Tosupportthis,dimeric
flavan-3-ols with the alternative C6ꢁ-C6 IFLs were hypothesized
in the products of PPO catalyzed or peroxidase mediated (+)-
catechin oxidation.[13]
Among the three trimeric flavan-3-ols, peak 6 had the same
[M−H]− molecularionatm/z 865asthenaturalB-typeprocyanidin
trimer (or C-type procyanidin), but had the completely different
fragment ions at m/z 847, 821, 803, 727, 713, 681, 577 and 425,
as shown in Fig. 2(a). The appearance of the fragment ions at m/z
727 and 681 and the absence of fragment ions at m/z 739 and
695, revealed that it might be an autoxidation trimer that had the
completely different structure from the natural B-type procyanidin
trimer. According to the report of Sun and Miller, the fragment
ions of B-type dehydrodicatechin at m/z 439, 425 and 393 were
formed by the retro-Diels–Alder (RDA) fragmentation on the C-
c
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Copyright ꢀ 2008 John Wiley & Sons, Ltd.
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F. He et al.
Table 1. Phenolic products of the autoxidation of flavan-3-ols detected by HPLC-MS/MS
Substrate
Peak No.
Product
Rt (min)
[M − H]− (fragment ions–MS2) (m/z)
(+)-Catechin
1
2
3
4
B-type dehydrodicatechin B11
Unknown
11.0
39.4
41.6
43.2
577 (533,439,425,393,269)
854 (792)
Unknown
628 (565,447)
741 (679)
Unknown
(−)-Epicatechin
5
6
A-type dehydrotricatechin
B-type dehydrotricatechin
(+)-Catechin
5.8
12.7
17.8
22.1
23.0
27.4
31.4
39.4
41.6
43.2
863 (725,711,699,575,425,411)
865 (727,713,681,577,425)
289 (245,205,151)
7
8
B-type dehydrodicatechin By
B-type dehydrodicatechin By
B-type dehydrodicatechin B22
A-type dehydrotricatechin
Unknown
577 (533,439,425,393,269)
577 (533,439,425,393,269)
577 (533,439,425,393,269)
863 (725,711,701,575,425,413)
854 (792)
9
10
11
2
3
Unknown
628 (565,447)
4
Unknown
741 (679)
(+)-Catechin and (−)-epicatechin
5
1
A-type dehydrotricatechin
B-type dehydrodicatechin B11
B-type dehydrodicatechin B12
B-type dehydrodicatechin By
B-type dehydrodicatechin B22
A-type dehydrotricatechin
Unknown
5.8
11.0
12.2
23.0
27.4
31.4
35.9
37.5
40.0
41.6
43.2
863 (725,711,699,575,425,411)
577 (533,439,425,393,269)
577 (533,439,425,393,269)
577 (533,439,425,393,269)
577 (533,439,425,393,269)
863 (725,711,701,575,425,413)
273 (229,187,139)
12
9
10
11
13
14
15
3
Unknown
591 (439,343,301,289,215)
591 (439,343,301,289,215)
628 (565,447)
Unknown
Unknown
4
Unknown
741 (679)
rings of the flavan-3-ol monomers.[14] The main fragment ions
of the potential autoxidation trimer also represented the similar
patterns of dissociation, because the m/z 727 ion to fragment
into m/z 439, 713 ion to generate into m/z 425, as well as m/z
681 ion into m/z 393 were all by losing a m/z 288 ion, which
corresponded to one monomeric flavan-3-ol by removing two
hydrogen atoms. These results indicated that flavan-3-ol units
in this trimer were linked with the same kind of IFL identified
in B-type dehydrodicatechin. Meanwhile, the appearance of the
fragmentionsatm/z 577and425suggestedthepossibilitythatthe
C6ꢁ-C8 IFL in the autoxidation trimer could be dissociated by the
striking effect (QM cleavage of IFL) to form its corresponding
dimers. Similar (+)-catechin trimers were synthesized in the
peroxidase-catalyzed and strawberry PPO catalyzed oxidation of
(+)-catechin, which were analyzed by reversed-phase (RP) HPLC
without coupling with MS.[5] In the present experiment, it was
the first time, to our knowledge, to synthesize and identify the
autoxidation trimeric flavan-3-ols of this unnatural type from the
autoxidationofflavan-3-ols,andthistrimerwasidentifiedasB-type
dehydrotricatechin. Scheme 2 displayed the detailed structure of
the [M − H]− molecular ion of the B-type dehydrotricatechin and
its partial fragment ions.
typical A-type procyanidin trimers, as shown in Fig. 2(b) and (c).
The appearance of the common fragment ions at m/z 845, 819,
801, 725, 711, 575 and 425 showed their similar but different
configurations with the identified B-type dehydrotricatechin,
suggesting the existence of additional IFL. According to the report
of Guyot et al., B-type dehydrodicatechin was easily oxidized into
A-type dehydrodicatechin by eliminating two hydrogen atoms
and forming an additional C–O–C IFL between two aromatic
rings, especially between the B-ring of the upper unit and
the A-ring of the lower unit.[12] On the other hand, if the
additional ether-type IFL (C–O–C bond) had no influence on
the C1ꢁ position on the B-ring of the first flavan-3-ol unit and
there was no more additional oxygen atom linked to the same
aromatic ring, the sixth main fragment ion should be at m/z
679 (681 minus 2), according to the dissociation rule we found
in the B-type dehydrotricatechin. However, we did not detect
this fragment ion in their – MS2 information, but obtained new
fragment ions at m/z 699 or 701. Therefore, according to the
basic structures of A-type dehydrodicatechins obtained by Guyot
et al., we hypothesized the detailed structures of the [M − H]−
molecular ions and partial fragment ions of the two trimeric
flavan-3-ols, and identified them as A-type dehydrotricatechins,
as shown in Scheme 3 (peak 5) and Scheme 4 (peak 11).[12] As
for peak 5, we concluded that one additional ether-type IFL
was formed between the C1ꢁ position on the B-ring of the first
unit and C7 position on the A-ring of the second unit, which
underwent benzopyran rearrangements during the autoxidation
and formed a quinone-methide structure of these rings. For peak
11, besides the ether-type IFL we mentioned before, in the first
flavan-3-ol unit an additional ether-type IFL was formed and
The other two trimers of flavan-3-ols, peaks 5 and 11 had
the same [M − H]− molecular ion at m/z 863 as those of the
natural A-type procyanidin trimer, which had been found in
various plants, for example, several natural A-type procyanidin
trimers isolated from plums and cinnamons and identified by
Gu et al.[10] Interestingly, the two new trimers almost had the
same fragment ions, at m/z 845, 819, 801, 725, 711, 699/701, 575,
425 and 411/413, which were totally different from those of the
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MS/MS of autoxidation oligomers of flavan-3-ols
Figure 2. ESI-MS2 spectra of three trimeric flavan-3-ols produced in the autoxidation of (−)-epicatechin. (a) B-type dehydrotricatechin (peak 6 in Table 1);
(b) A-type dehydrotricatechin (peak 5 in Table 1); (c) A-type dehydrotricatechin (peak 11 in Table 1).
this IFL contacted the C3 position on the C-ring and the C3ꢁ
position on the B-ring. Thus, the B-type dehydrotricatechin was
considered as the most probable precursor of the two A-type
dehydrotricatechinswhichwereformedthroughfurtheroxidation
and subsequent intramolecular radical additions, and the different
formationmechanismsresultedintheirdifferentstructures, similar
but partially different fragment ions, and totally different retention
times.
in the autoxidation of (−)-epicatechin in the mildly acidic
model solution. Similar products were also formed after storage
of a (+)-catechin model medium (pH 5.7) for a long time
(30 days) by Callemien and Collin,[15] but were not present
in the autoxidation of (+)-catechin in our tests (24 h), which
indicated that (−)-epicatechin might be more easily to be
oxidated than its stereoisomer (+)-catechin. On the other
hand, no A-type dehydrodicatechins were synthesized in our
reaction, which was also different from previous reports of other
researchers.[16]
Therefore, we obtained three B-type dehydrodicatechins, one
B-type dehydrotricatechin and two A-type dehydrotricatechins
c
J. Mass. Spectrom. 2009, 44, 633–640
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F. He et al.
Scheme 2. Detailed structures of deprotonated B-type dehydrotricatechin (peak 6 in Table 1) and its partial fragment ions.
Scheme 3. Detailed structures of deprotonated A-type dehydrotricatechin (peak 5 in Table 1) and its partial fragment ions.
c
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MS/MS of autoxidation oligomers of flavan-3-ols
Scheme 4. Detailed structures of deprotonated A-type dehydrotricatechin (peak 11 in Table 1) and its partial fragment ions.
Autoxidation in the mixture containing (+)-catechin and (−)-
(the B-type procyanidin dimers at m/z 451, the B-type procyanidin
epicatechin
trimers at m/z 739, the A-type procyanidin trimers at m/z 737) in
complex samples. Although the mass spectrum data alone could
not provide us enough information about the exact structures of
these autoxidation oligomeric flavan-3-ols, MS/MS coupled with
reversed-phase HPLC provides a rapid and sensitive method for
their determination and identification.[10,14]
In the autoxidation mixtures of (+)-catechin and (−)-epicatechin
carried out in aqueous/methanol model solutions under mildly
acidic condition (pH 6.0), 11 products were detected besides
the residual substrates (+)-catechin and (−)-epicatechin, and the
buffer content MES (Fig. 1(c)), as summarized in Table 1.
Besides the B-type dehydrodicatechin B11 (peak 1) which was
also detected in the autoxidation of (+)-catechin, one unknown B-
type dehydrodicatechin By (peak 9) and B-type dehydrodicatechin
B22 (peak 10) that also existed in the autoxidation of (−)-
epicatechin, the typical B-type dehydrodicatechin B12 (peak 12)
which was composed of one (+)-catechin unit and one (−)-
epicatechin unit was obtained. In addition, the two new A-type
dehydrotricatechins (peaks 5 and 11) were also detected but no
B-type dehydrotricatechin was obtained. However, besides peaks
3 and 4 that were also formed in the autoxidation of either
(+)-catechin or (−)-epicatechin, three new products of unknown
species were also detected, including the isomer peaks 14 and 15,
and a rather smaller compound peak 13.
Acknowledgements
This research was supported by National Natural Science Founda-
tion of China (grant No.30671467 to C.-Q.D).
References
[1] L. Pourcel, J.-M. Routaboul, V. Cheynier, L. Lepiniec, I. Debeaujon.
Flavonoid oxidation in plants: from biochemical properties to
physiological functions. Trends in Plant Science 2007, 12, 29.
[2] H. Peleg,K. Gacon,P. Schlich,A. C. Noble.Bitternessandastringency
of flavan-3-ol monomers, dimers and trimers. Journal of the Science
of Food and Agriculture 1999, 79, 1123.
[3] J. E. O’Connell, P. F. Fox. Significance and applications of phenolic
compounds in the production and quality of milk and dairy
products: a review. International Dairy Journal 2001, 11, 103.
[4] P. M. Aron, J. A. Kennedy. Flavan-3-ols: Nature, occurrence and
biological activity. Molecular Nutrition and Food Research 2008,
52, 79.
[5] M. Lo´pez-Serrano, A. R. Barcelo´. Comparative study of the products
of the peroxidase-catalyzed and the polyphenoloxidase-catalyzed
(+)-catechin oxidation. Their possible implications in strawberry
(Fragaria × ananassa) browning reactions. Journal of Agricultural
and Food Chemistry 2002, 50, 1218.
[6] J. Oszmianski, V. Cheynier, M. Moutounet. Iron-catalyzed oxidation
of (+)-catechin in model systems. Journal of Agricultural and Food
Chemistry 1996, 44, 1712.
[7] D. E. Hathway, J. W. T. Seakins. Autoxidation of polyphenols. Part
III. Autoxidation in neutral aqueous solution of flavans related to
catechin. Journal of the Chemical Society 1957, 300, 1562.
[8] K. Weinges, W. Ebert, D. Huthwelker, H. Mattauch, J. Perner.
Oxidative coupling of phenols, II. structure and mechanism of
Conclusions
Under the mildly acidic condition (pH 6.0), flavan-3-ols trended to
condense with each other to synthesize a series of unnatural
oligomers in the autoxidation reactions. With the help of
HPLC-MS/MS, we could identify these oligomers as the B-
type dehydrodicatechins, the B-type dehydrotricatechins and
the A-type dehydrotricatechins, respectively, and speculated
the detailed structures of their [M − H]− molecular ions and
partial fragment ions. Further more, the specific fragment
ions could be used to rapidly distinguish these autoxidation
oligomers (the B-type dehydrodicatechins at m/z 393, the B-type
dehydrotricatechins at m/z 681, the A-type dehydrotricatechins
at m/z 725) from their corresponding natural procyanidin isomers
c
J. Mass. Spectrom. 2009, 44, 633–640
Copyright ꢀ 2008 John Wiley & Sons, Ltd.
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F. He et al.
the formation of dehydrodicatechin A. Justus Liebigs Annalen der
Chemie 1969, 726, 114.
catalyzed by grape polyphenoloxidase. Phytochemistry 1996, 42,
1279.
[9] S. Guyot, V. Cheynier, J. M. Souquet, M. Moutounet. Influence of pH
on the enzymatic oxidation of (+)-catechin in model systems.
Journal of Agricultural and Food Chemistry 1995, 43, 2458.
[10] L. Gu, M. A. Kelm, J. F. Hammerstone, Z. Zhang, G. Beecher,
[13] J. Sun, F. Liang, Y. Bin, P. Li, C. Duan. Screening non-colored
phenolics in red wines usingliquid chromatography/ultraviolet and
massspectrometry/massspectrometrylibraries.Molecules2007,12,
679.
J. Holden,
D. Haytowitz,
R. L. Prior.
Liquid
chromato-
[14] W. Sun, J. M. Miller. Tandem mass spectrometry of the B-type
procyanidins in wine and B-type dehydrodicatechins in an
autoxidation mixture of (+)-catechin and (−)-epicatechin. Journal
of Mass Spectrometry 2003, 38, 438.
graphic/electrospray ionization mass spectrometric studies
of proanthocyanidins in foods. Journal of Mass Spectrometry 2003,
38, 1272.
[11] I. Tarascou, K. Barathieu, C. Simon, M. A. Ducasse, Y. Andre´,
E. Fouquet, E. J. Dufourc, V. Freitas, M. Laguerre, I. Pianet. A 3D
structural and conformational study of procyanidin dimers in
water and hydro-alcoholic media as viewed by NMR and molecular
modeling. Magnetic Resonance in Chemistry 2006, 44, 868.
[12] S. Guyot, J. Vercauteren, V. Cheynier. Structural determination of
colourless and yellow dimers resulting from (+)-catechin coupling
[15] D. Callemien, S. Collin. Involvement of flavanoids in beer color
instability during storage. JournalofAgriculturalandFoodChemistry
2007, 55, 9066.
[16] M. Lopez-Serrano, R. Barcelo. Reversed-phase and size-exclusion
chromatography as useful tools in the resolution of
peroxidase-mediated (+)-catechin oxidation products. Journal of
Chromatography A 2001, 919, 267.
c
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