Sequencing of red wine proanthocyanidins by UHPLC-ESI-Q-ToF

Phenolic compounds are considered as secondary metabolites and are widespread in the plant kingdom [1, 2]. These compounds are present in vegetables [3], fruits [4], tea [5] and red wine [6-8]. They are known for their oxidative defense [9], their ability to reduce certain cancers [10, 11], their preventive activity against infectious [12] and degenerative diseases [13,14]. Among these phenolic compounds, the proanthocyanidins (PAs) or flavan-3-ols represent a significant family and they play an important role during wine making [15] and red wine tasting [16]. Four monomeric units [17, 18] are present in the grape and red wine: (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC) and (-)-epicatechin-3-O-gallate (ECG) (Figure 1). These monomers give rise to the formation of oligomers and polymers via an interflavan bond between C4 of the top unit and C6 or C8 [4, 19] of the lower unit and sometimes an additional ether bond between C2 of the top unit and C5 or C7 of the lower unit [20, 21]. (-)-epicatechin (EC), (+)-catechin (C) and (-)-epicatechin-3-O-gallate (ECG) are mainly located in grape seeds, whereas the monomeric unit (-)-epigallocatechin (EGC) is only present in grape skins [22, 23]. These compounds present in red wine are involved in the astringency phenomenon [16, 17], the bitterness, the body [24], the wine aging [25] and the organoleptic properties [26]. These proanthocyanidins have been studied by analytical method such as high-performance liquid chromatography (HPLC) [27], mass spectrometry coupled with UHPLC system [28], and nuclear magnetic resonance (NMR) [29]. In the current study, we first describe the theoretical possibilities to form oligomers with A and B-type interflavan bond. In a second part, we describe specific fragmentation pathways allowing the sequencing of proanthocyanidins in red wine using a UHPLC-ESI-Q-ToF. JOURNAL OF APPLIED BIOANALYSIS, Apr. 2015, p. 46-54. http://dx.doi.org/10.17145/jab.15.009 (ISSN 2405-710X) Vol. 1, No. 2

In the current study, we first describe the theoretical possibilities to form oligomers with A and B-type interflavan bond.In a second part, we describe specific fragmentation pathways allowing the sequencing of proanthocyanidins in red wine using a UHPLC-ESI-Q-ToF.

Chemicals
All chemicals were of analytical reagent grade.Acetonitrile was purchased from Fisher Scientific (Waltham, MA, USA) and formic acid from Sigma Aldrich (St Louis, MO, USA).Deionized water was purified with a MilliQ water system (Millipore, Bedford, MA).Red wine analyzed was from Okanagan Valley (VQA, Okanagan Valley) 2010 Merlot, barrel sample, sampled in May 2010 and was kept in the freezer at -20°C before analysis.Red wine sample was just filtered with PTFE membrane (VWR).

UHPLC and ESI-MS conditions
Analyses of red wine were carried out using UHPLC-ESI-Q-ToF (Agilent 6530, Series Accurate Mass Q/TOF MS, Agilent Technologies, Santa Clara, CA) in negative mode.The UHPLC system (Agilent 1290 Series, Agilent Technologies, Santa Clara, CA) equipped with an autosampler, a vacuum degaser, a binary pump, a quaternary pump, a thermostated column department and a diode-array detector.Reversed phase UHPLC analyses were performed using a C18 Column (Zorbax SB, 2.1 x 150 mm, 1.8 µm, Agilent Technologies, Santa Clara, CA) maintained at 30ºC, and the flow rate was 0.4 mL.min-1.The binary mobile phase consisted of water with 0.1% formic acid as mobile phase A and acetonitrile with 0.1% formic acid as mobile phase B. The elution conditions were as follows: 0-8 min, 5% B isocratic; 8-32 min, 5-20% B; 32-40 min, 20-100% B; 40-44 min, 100% B isocratic; 44-48 min, 5% B, and followed by two minutes to re-equilibrate the column before the next run.Injection volume was 2 µL with the DAD detector set to an absorbance wavelength of 280 nm.The UHPLC system was connected to an ESI-Q-TOF.This instrument was worked in Extended Dynamic Range of 2 GHz (m/z 3200 Th) high-resolution mode.The parameters of detection were performed as follows: drying gas (N 2 ) flow rate, 10 L.min -1 ; sheath gas temperature 400 ºC; nebulizer pressure 25 psig; drying gas temperature 325 ºC; sheath gas flow 12 L.min-1; capillary voltage 3500 V; fragmentor voltage150 V; skimmer 65 V.The collision energy was set at 15 eV to 35 eV following the compounds targeted.All analyses were performed in negative mode, which is more sensible than the positive mode.Indeed, in comparison to the positive mode where sodium or potassium salts adducts can be detected, the negative mode produces cleaner spectra without those adducts [30].Each chromatogram of targeted compounds was obtained by extracted ion current (EIC) in MS/MS mode.

Results
Theoretical models for possible structures for Proanthocyanidins in red wine By using combinations, considering the number of units and the different ways these units can be linked together, a counting of the possible polymers can be done in order to depict the complexity of the wine matrix.The four monomeric units ((+)-catechin, (-)-epicatechin, (-)-epigallocatechin and (-)-epicatechin-3-O-gallate) lead to the polymer production.The polymerization occurs via the formation of interflavan linkage.The most common linkage is the B-type linkage between the C4 of the top unit and the C8 of the lower unit (noted C4→C8) or between the C4 of the top unit and the C6 of the lower unit (C4→C6).An additional ether bond between the C2 of the top unit and the C7 or C5 of the lower unit (C2-O-C7 or C2-O-C5) can occur and the linkage is then categorized as A-Type.In red wine the dimer A2 (two epicatechin linked with C4→C8 interflavan bond and C2-O-C7 ether bond) has been identified by Vivas and Glories [31].The main oligomers (dimers to pentamers) of A-Type characterized, are linked by C4→C8 bond and an ether bond between C2-O-C7 in grape seeds, plums, cranberries.The combination C4→C8 and C2-O-C5 is not considered, the interflavan linkage C4→C8 cannot create an ether bond C2-O-C5 because the atoms are too far apart.Considering B-type and A-type linkages, five possibilities exist to bond a unit to the next one: C4→C6; C4→C8; C4→C8 and C2-O-C7; C4→C6 and C2-O-C7; C4→C6 and C2-O-C5.Taking into consideration the four monomeric units and the five possible linkages, the theoretical number of different structures for a given number of monomeric unit can be determined.The table 1 shows the results up to the heptamer (DP7).

Theoretical fragmentation in MS/MS for B-Type dimers
The targeted MS/MS mode allowed us to obtain additional information about the proanthocyanidin structures.For all compounds studied theoretical fragmentation pathways were established, the specific fragments provide a unique signature that allows a better characterization and identification.However (-)-epicatechin (EC)  The same argument can be applied for the compounds including one EGC unit, the ion at m/z = 167.035and the ion at m/z = 441.0827are produced after the RDA cleavages.The theoretical fragmentation pathways established can be used to characterize the connection sequence of proanthocyanidins with specific signature.The theoretical fragmentation pathways and specific fragments of these B-type dimers were used to identify them in red wine (Table 2).

Identification of B-type dimers in red wine
According to Table 1 presenting the different possibilities for B-Type dimers, thirty-two theoretical combinations can be detected in red wine.Due to high similarity in term of structure, the only variation being the spatial

Theoretical fragmentation in MS/MS for A-Type dimers
Compared to the B-type dimers, A-type dimers follow the same the fragmentation pathway, the specific ions produced will differ only by 2 Da.

Identification of A-type dimers in red wine
According to the Table 1, thirty-six theoretical A-type dimers can be detected in red wine.For the EC(C)→ EGC series four compounds were observed: (1'), (2'), (3') and (4') (Table 4).Fragment ion at m/z = 465.0827can be generated from HRF1, the complementary ion was a loss of phloroglucinol.The cleavage of interflavan bond (QM) led to the formation of fragments at m/z = 305.0667and then followed by a loss of hydroxyl group to give rise the ion at m/z = 289.718.
The compounds with EC(C)→EGC sequences were detected and characterized: compounds ( 11), ( 12), ( 13) and ( 14).Two specific fragmentations, QM and HRF2 fission, allow the discrimination of EC(C)→EGC to EGC→EC(C) dimers.By targeted MS/MS the QM produced the fragment ions at m/z = 305.0675and m/z = 287.0561that are specific to EC(C)→EGC dimer.Compounds ( 15) and ( 16) were EGC→EGC dimers.The fragment ions at m/z = 483.0962and m/z = 125.0215can be attributed to the HRF1 (upper unit cleavage).Furthermore the ions at m/z = 441.0369and 167.0369 can be produced from the RDA (top and bottom unit).The QM fragmentation can also explain the occurrence of the fragment ions at m/z = 303.0482and 305.0664.Compounds ( 17), ( 18) and ( 19) correspond to EC(C)→ECG →ECG and ECG→ECG were not detected in red wine.Yet our sample was simply filtered, without prefractionation, these compounds could be present under the limit of detection of the spectrometer.Furthermore, in grape and red wine, the epicatechin-3-O-gallate unit is mainly present at the terminal position due to the steric effect.
Concerning A-type dimers the specific fragments obtained were in accordance with the literature [34].Twelve compounds seem to be in agreement with the theoretical fragmentation pathways established.For EC(C)→ EC(C) analogues, five compounds can be observed and four compounds for EC(C)→EGC series.These results have demonstrated the possibility of sequencing the dimers of A and B type.Thirty-two dimers can be identified in accordance with their specific fragmentation pathways.This method can be adapted to characterize the trimers present in red wine.However as we can see with the mathematical relationship, two hundred fifty-six different trimers could exist for B-type linkage only.The detection m/z = 617.0937.The fragment ion at m/z = 441.0827indicated an interflavan bond cleavage for this dimer.

Discussion
The first goal of this study was to enumerate all oligomers of proanthocyanidins thanks to mathematical relationships and then to detect them by mass spectrometry directly from a sample of wine.Regarding dimers, thirty-two theoretical combinations can be obtained for B-type.The use of UHPLC-ESI-Q-ToF and the theoretical fragmentation pathways allowed us to distinguish twenty compounds.For the EC(C)→EC(C) series, eight theoretical compounds were detected, the MS/MS data allowed their total identification.However, the catechin and epicatechin units cannot be discriminated because they only differ by one stereo center in position C3.The use of commercial standards could allow their identification.For the dimer containing gallate unit (ECG), the series ECG→EC(C), EGC  of trimer is possible but it represents a long and fastidious work and the complexity increases exponentially with the degree of polymerization.So far, liquid chromatography coupled with tandem mass spectrometry is the best technique to detect those compounds directly from filtered wine without further preparation.But looking at the theoretical number of molecules it is obvious that we cannot detect everything.

Conclusion
The partial identification of dimers with B and A-type linkage in red wine using an UHPLC-ESI-Q-ToF without sample preparation was reported for the first time.This work has also showed the complexity of oligomers present in red wine with the mathematical relationship established.The targeted MS/MS, high resolution and fragmentation pathways allowed the distinction of twenty among thirty-two possible B-type dimers and twelve among thirty-six A-type dimers.For the A-type dimers, the interflavan bond seems to be an ether bond between C2 and C7 according to the fragment ions detected.The sequencing of these compounds was in accordance with their specific signatures.The results obtained showed the efficiency of the mass spectrometer used.To confirm the sequencing, the same method could be developed with a triple quadrupole or an ion trap.Furthermore, the separation of these compounds could be considered to study the organoleptic properties of A-type dimers.

Figure 1 .
Figure 1.Monomeric units and dimers (A and B-type).

Figure 2 .
Figure 2. Theoretical fragmentation pathway for B-Type dimer with specific cleavages unit.If the sequence is EGC→EC(C) after the QM, the cleavage generates the ion at m/z = 439.0671identified as ECG unit and the monomeric unit EC(C) at m/z = 289.0718(bottom unit).For the other sequences EGC→EC(C), EC(C)→EGC, EGC→ECG and EC-G→EGC, the quinone methide fragmentation are specific, when EGC, EC(C), and ECG are bottom unit the fragmentation pattern gives the exact mass of the monomeric unit.For the compounds containing the monomeric unit EC(C), the RDA (top and bottom unit) produces the ion at m/z = 151.0401and the ion at m/z = 425.0878.The m/z = 137.0244indicates another specific fragment for the monomeric unit EC(C) resulting from a RDA or OC9/C2C3 cleavage.
').The fragment ions at m/z = 289.0718and m/z = 285.0405resulted from the quinone methide cleavage.Neutral loss of phloroglucinol (m/z = 125.0244)and fragment ion at m/z = 449.0878indicated a HRF1 on the upper unit.The retro Diels Alder cleavages (RDA1 and RDA2) produced two specific ions at m/z = 423.0722and m/z = 425.0878respectively.One peak (10') was assigned to EGC→EGC A-type dimer.The MS/MS spectra obtained showed the ions at m/z = 305.0667and m/z = 301.0354characteristic from interflavan bond cleavage.Another specific fragmentation was the RDA1 with the ions at m/z = 439.0671.Compound (11') can be EC(C)→ECG A-type dimer.The quinone methide cleavage led to the formation of two specific fragments at m/z = 441.0827and m/z = 285.0405.The ion m/z = 441.0827 is characteristic when the gallate unit is bottom unit.Neutral loss of gallate unit produced the dimer at m/z= 577.1195.Compound (12') appears to be EGC→ECG dimer.The heterocyclic ring fission give rise to m/z = 427.0671and arrangement due to stereochemistry and the position of the interflavan bond, proanthocyanidins dimers tend to have identical masses and fragments.For instance, in Table3, the dimer EC(C)→ECG had three different retention times corresponding to three of the four possibilities: EC→ECG and C→ECG, which can be linked in C4/C6 or C4/C8.The theoretical fragmentation pathway of these four molecules generates the same specific fragments.For the EC(C) dimer series the specific fragmentation pathway allowed the characterization of eight compounds.For the dimer EC(C)→EC(C), compound 1 (m/z = 577.1351,rt = 12.726), the fragment ion at m/z = 559.1214,was a loss of water from the precursor ion.Neutral loss of phloroglucinol and fragment ion at m/z = 451.1022indicated a HRF on the upper unit.Specific ions at m/z= 287.0555 and m/z = 289.0718were detected, thereby indicating a QM cleavage of the interflavan bond.For the RDA fragmentation (bottom and top unit), the ions at m/z = 425.0801and m/z = 151.0398were produced.The formation of the fragment at m/z = 401.0779indicated a loss of water after RDA (upper and lower unit) cleavage produced from m/z = 425.0881.

Table 1 .
All possibilities to form proanthocyanidins.
[30,32,33]techin (C) are indistinguishable because they only differ by the stereochemistry of hydroxyl group in position 3 on ring C, the abbreviation EC(C) will be used to describe it.The main cleavages of proanthocyanidins are: retro-Diels Alder (RDA), heterocyclic ring fission (HRF), quinone methide or interflavan bond cleavage (QM) and sometimes benzofuran forming fission (BFF)[30,32,33](Figure2).For each compound a targeted MS/MS mode was developed with optimized collision energy accordingly to the mass of the molecule.Figure2describes the different fragmentation patterns occurring for B-type dimers.Using these fragmentation patterns, the sequence of the proanthocyanidin can be resolved.For instance, a B-type dimer of (epi)catechin and epicatechin-3-O-gallate presmass, except for (-)-epicatechin and (+)-catechin series.With EC(C)→ECG, the quinone methide produces the ion at m/z = 287.0561,specific fragments when EC(C) is top unit, and m/z = 441.0827for the ECG bottom

Table 2 .
Specific fragments obtained in negative mode for B-type dimers

Table 3 .
Proanthocyanidin B-Type dimers detected and identified by mass spectrometry in red wine sample.

Table 4 .
Proanthocyanidin A-Type dimers detected and identified by mass spectrometry in red wine sample.