Isotopic Exchange HPLC-HRMS/MS Applied to Cyclic Proanthocyanidins in Wine and Cranberries
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Cyclic B-type proanthocyanidins in red wines and grapes have been discovered recently. However, proanthocyanidins of a different chemical structure (non-cyclic A-type proanthocyanidins) already known to be present in cranberries and wine possess an identical theoretical mass. As a matter of fact, the retention times and the MS/MS fragmentations found for the proposed novel cyclic B-type tetrameric proanthocyanidin in red wine and the known tetrameric proanthocyanidin in a cranberry extract are herein shown to be identical. Thus, hydrogen/deuterium (H/D) exchange was applied to HPLC-HRMS/MS to confirm the actual chemical structure of the new oligomeric proanthocyanidins. The comparison of the results in water and deuterium oxide and between wine and cranberry extract indicates that the cyclic B-type tetrameric proanthocyanidin is the actual constituent of the recently proposed novel tetrameric species ([C60H49O24]+, m/z 1153.2608). Surprisingly, the same compound was also identified as the main tetrameric proanthocyanidin in cranberries. Finally, a totally new cyclic B-type hexameric proanthocyanidin ([C90H73O36]+, m/z 1729.3876) belonging to this novel class was identified for the first time in red wine.
KeywordsCyclic proanthocyanidins Hydrogen/deuterium exchange High-resolution mass spectrometry Wine Cranberries Cyclic B-type hexameric proanthocyanidin
Proanthocyanidins (PAC) constitute a class of oligomeric compounds present in grape skin and seeds composed of flavan-3-ol ((epi)catechin) units [1, 2, 3, 4]. In recent pioneering reports, the presence of tetrameric and pentameric novel cyclic PAC (therein referred as crown proanthocyanidins) in red wines from the Bordeaux region was proposed for the first time [5, 6]. MS and NMR characterizations of the isolated molecules from purified extracts were provided. Despite the thorough characterization performed, this observation (the cyclic ‘crown’ structure of these PAC) was affected by an unforeseen ambiguity: the proposed novel cyclic B-type PAC share the same elemental composition with an already known class of (non-cyclic) PAC (seldom observed in wine) containing the additional A-type O-C linkage, usually present between the n and n-1 monomers in a n-mer [7, 8]. An example of a compound of that non-cyclic PAC class in red wine is procyanidin A2 . In addition, this A-type PAC class up to the tetramer was reported to be abundant in cranberries . The proposed novel cyclic B-type PAC and the non-cyclic A-type PAC possess identical elemental compositions (see the Results section for details). Therefore, the application of mass spectrometry for directly clarifying the actual structure of the new cyclic oligomers in wine may not be straightforward. MS n methods may also yield insufficient results since the A-type linkage can be broken during MS/MS fragmentation leaving only the B-type linkage [7, 8]. In this respect, a direct approach supporting the structural NMR characterization would be helpful [5, 11]. This approach may be achieved by devising a chemical modification able to differentiate between the two different chemical bond arrangements of the two PAC classes (cyclic B-type and non-cyclic A-type).
Among the most exploited derivatization approaches [12, 13, 14, 15], hydrogen/deuterium (H/D) exchange offers a valuable option for structural resolution whenever exchangeable protons are present. Moreover, the H/D exchange rates influence conformational mobility, hydrogen bonding strength, and solvent accessibility of the molecule studied [16, 17, 18, 19, 20]. Nonetheless, this modification is completely reversible, and care must be applied to obtain a complete deuteration. Accordingly, HPLC methods have been modified for fully profiting from this transformation, for example by employing deuterium oxide as replacement of the water phase in HPLC-MS gradient methods . A critical step is the equilibration time for a complete H/D exchange, in particular for proteins, which is influenced by the substrate, the solution pH, and the temperature . H/D exchange was infrequently but successfully applied to wine extracts to characterize anthocyanins and pyranoanthocyanins, demonstrating how the H/D exchange rate followed an apparent first order kinetic independent from the solution pH .
In this report, H/D isotopic exchange was applied to confirm the B-type cyclic structure for a tetramer PAC recently proposed [5, 6] possessing the same theoretical mass and elemental composition of the non-cyclic A-type tetrameric proanthocyanidin. The different number of exchangeable protons between the novel cyclic oligomers and their non-cyclic A-type analogs implies that a complete H/D exchange should remove the isobaric property and reveal the actual structure. For comparison, the same procedure was applied on a concentrated cranberry extract, which was shown to possess more of the A-type PAC . Therefore, a direct comparison of the HPLC-HRMS/MS spectra could add valuable information. In addition, the presence of a novel cyclic B-type hexameric PAC is proposed in red wine.
LC-MS grade solvents and additives were purchased from Sigma Aldrich Srl (Milano, Italy). D2O used was 99.9% of D atoms. Wine samples (Lagrein, 2016 vintage) were donated by a local winery (Kellerei Bozen, Bolzano, Italy). Dried cranberries were purchased from a local market.
No preliminary extraction was applied to the wine samples. Dried cranberries were initially extracted according to a published protocol . Briefly, 15 g of dried and refrigerated cranberries were extracted with 135 mL of acetone/water/acetic acid (70:29.5:0.5 v/v). The mixture was sonicated (99 W for 5 min at 50 oC), kept at 50 oC for 30 min, vortexed, and centrifuged at 3300 rpm. All samples (wine and cranberries) were concentrated at low pressure (9–10 mbar) followed by 30 min of gentle N2 flux; then they were reconstituted to a final concentration 10 to 30 times higher than the starting sample. When H/D exchange was performed, the samples were first dried and then reconstituted 3–4 times in pure deuterium oxide before fluxing N2 and reconstituting in the deuterated mobile phase A. The samples were always filtered (0.2 μm, regenerate cellulose) before injection.
The HPLC-HRMS/MS method applied was adapted from a published report . The HPLC-HRMS system used consisted of a QExactive HRMS instrument (Thermo Fisher Scientific, Rodano, Milano, Italy) coupled to an Agilent 1260 HPLC (Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Milano, Italy) with a 16-channel DAD detector. The separation was performed at a flow rate of 1 mL·min-1 with a ODS Hypersyl C18 LC column (125 mm × 4.6 mm i.d., 5 μm, Thermo Fisher Scientific) protected with a HPLC pre-column filter (ODS Hypersil, 5 μm pore size, 10 × 4 mm drop-in guards, Thermo Fisher Scientific). A contact closure electronic board provided synchronization of the HPLC with the mass spectrometer detector. The mobile phase consisted of a combination of solvent A (0.1% v/v formic acid in 0.02 mol·L-1 ammonium formate in water or 0.1% v/v deuterated formic acid in 0.02 mol·L-1 fully deuterated ammonium formate in deuterium oxide) and solvent B (0.1% v/v formic acid in saturated ammonium formate acetonitrile or 0.1% v/v deuterated formic acid in saturated fully deuterated ammonium formate acetonitrile, LC-MS grade). The gradient was set as follows: from 5% B at 0 min to 25% B at 21 min, then to 95% B at 22 min to 27 min, to 5% at 28 min, followed by a re-equilibration step (5% B) at 32 to 35 min. The DAD spectra were recorded from 210 to 600 nm and provided real-time monitoring at 280, 320, 365, 420, and 520 nm (+/– 4 nm). A post-column flow splitter was used to feed both analyzers in parallel (DAD and HRMS) at a fixed ratio. For full MS analysis, the mass spectrometer was operated in positive ionization mode using the following conditions for the HESI ionization source: sheath gas at 20 (arbitrary units), aux gas at 5 (arbitrary units), aux temperature 250 °C, spray voltage at +3.00 kV, capillary temperature at 320 °C, and rf S-lens at 70. The mass range selected was from 500 to 2000 m/z with a full MS set resolution of 70,000 (@200 m/z), AGC target at 3e6, max. injection time of 300 ms. To maximize the sensitivity, data-dependent LC-MS/MS experiments were run on the N2 concentrated samples and on a selection of 35 ions only (manually specified in the inclusion list): full-MS parameters were kept as shown, MS/MS AGC target 106, max. injection time 300, FT-MS set resolution 35,000, loop count 5, isolation window 2 m/z, normalized collision energy 15 eV. For data-dependent settings: minimum AGC target 3.103, apex trigger 2 to 8 s, charge exclusion 3–8 and higher, dynamic exclusion 3 s, “if idle” tool set to “do not pick others.” LC-MS/MS experiments were also tested in negative ionization mode (spray voltage –3.50 kV) but with lower response factor; therefore, they are not discussed any further. Lock masses were consistently employed to correct mass deviations across the Full MS acquisition range throughout the experiments. When deuterium oxide was employed, the lock masses were modified accordingly into the main instrument method. The HPLC-DAD data were collected and analyzed by OpenLab software whereas the MS data and results were collected and analyzed by Xcalibur 3.1 software. MS/MS fragmentation interpretation was performed on the basis of comparison with the cited literature, and it was aided by Mass Frontier 7.0 software.
Results and Discussion
With respect to the cyclic analogs, the linear ones showed a higher level of fragmentation, in particular through the QM mechanism. In all spectra, m/z 577.131 was the most intense peak, whereas in the cyclic forms the pseudo-molecular ions (m/z 1729.381, 1441.317, and 1153.259 for CHP, CPP, and CTP, respectively) were the highest. We could consider this as an interesting marker of distinction between the cyclic and the linear forms. However, also RDA (with or without further water loss) contributed in the MS/MS of the linear forms (e.g., m/z 1291.281, 1273.279, 1003.225, 715.161, and the three main m/z 695.136, 427.101, 425.085, and 409.090). HFR contributed as well (e.g., m/z 1317.816 and 451.100). However, the most remarkable difference between cyclic and linear species is the preference towards the trimeric and dimeric fragments. For the cyclic PAC, m/z 865.197 and 577.131 are the main (and only) trimeric and dimeric peaks, whereas for the linear PAC, m/z 867.209 and 579.147 also contribute. Considering that these fragments are produced via the reported QM mechanism (in which the monomer released from the main chain is virtually oxidized), we could infer that this difference is directly related to the cyclic nature of CHP, CPP, and CTP. In fact, the presence of the additional head-tail C–C bond forced the main chain also to be in a –2[H] state, and this may generate the –2 Da difference in certain peaks for the cyclic forms. The other main differences, as already stated, are the fragmentation preferences: (1) trimeric and tetrameric forms are almost negligible in the cyclic pentamer and hexamer spectra; (2) the much higher fragmentation level of the linear PAC with respect to the cyclic species.
Theoretical masses (pseudo-molecular ion, [M H +H]+ and [M D +D]+) and elemental compositions for native and deuterated tetramers
Elemental composition (ion)
Theoretical mass (m/z)
B-type cyclic tetramer
B-type non-cyclic tetramer
A-type non-cyclic tetramer
B-type cyclic tetramer-d20
B-type CTP-d 20
B-type non-cyclic tetramer-d20
B-type LTP-d 20
A-type non-cyclic tetramer-d19
A-type LTP-d 19
In this report, only the tetrameric species could be investigated any further; low concentrations and a decreased sensitivity with deuterium oxide prevented the analysis of the less abundant higher oligomers.
The A-linkage (in Figure 3a) is indicated with an arrow. Likewise, the B-linkage that differentiates the proposed novel cyclic B-type PAC (Figure 3b) from the non-cyclic B-type PAC (Figure 3c) is indicated with an arrow.
The species in Figure 3a and b are isobaric. However, as aforementioned, all the O–H protons should undergo H/D isotopic exchange, whereas all the C–H protons should not. Therefore, with an A-linkage (A-type LTP, Figure 3a) one exchangeable proton (O–H) is lost with respect to the B-type LTP species (Figure 3c) since the A-type C–O bond is in its place. Conversely, no exchangeable proton is lost with a new C–C B-linkage (B-type CTP, Figure 3b) in place of two C–H bonds. As a result, the two deuterated species (in Figure 3d and e, respectively) should not be isobaric anymore, as shown in Table 1.
The retention times (of the cyclic and the linear oligomers alike) in deuterium oxide were delayed for over 60 s with respect to water (in Figure 1), which was reasonable since deuterium oxide exerts a weaker hydrogen bonding than does water. The retention time differences upon H/D exchange were already reported as dependent upon the specific compound eluted .
A loss in relative abundance was observed with deuterium. Nonetheless, these preliminary results suggest that the presence of the proposed cyclic compounds only (and not the non-cyclic A-type LTP) would properly explain the results for the tetramer. Traces of a co-eluting species compatible with the A-type LTP were indeed observed (Figure 4). However, this was due to a contribution of an overlapping m+2 isotopic peak of a yet unidentified m/z 1171.536 (z = +2) eluting at 5.4 min (EIC not shown). Nonetheless, the relative abundance of the peak at m/z 1173 is negligible (less than 2% of B-type CTP). The MS/MS fragmentation pattern was also recorded. Notably, all peaks were shifted with respect to the corresponding spectrum in water (Figure 2) after H/D exchange. These values are indicated in italics in the spectrum. MS/MS spectrum analysis has been performed in the spectrum in H2O and reported in the discussion (and in Figure 2). The fragmentation pattern for deuterated CTP reported in Figure 4 is completely analogous to the one reported in Figure 2-A. The pseudo-molecular ion peak at m/z 1174.387 showed a similar level of fragmentation with respect to the native (hydrogenated) species. The fragmentation mechanisms were altogether the same. Just as for the cyclic tetramer of Figure 2, the trimer, dimer, and monomer fragments were also produced here via the QM mechanism (m/z 881.294, 588.201, and 295.107, respectively). Moreover, RDA mechanism accounted for the losses of 3,4-dihydroxyphenylacetaldehyde-d3 with or without the further loss of deuterated water (m/z 1019.321, 706.204, and 415.127). Notably, looking at the progressive loss of monomer units of Figure 4, it is possible to notice a difference of exactly one monomer unit containing five exchangeable protons. Taking into account the difference given by the deuterium needed for providing the ionization, exactly 20 u are added to the tetramer pseudo-molecular ion, 15 u to the trimeric form, 10 u to the dimeric form, and 5 u to the monomeric unit.
The MS/MS at 3.8 ± 0.1 min (7-A) matches instead most of the ion fragments of m/z 1155.27 MS/MS (7-C, B-type LTP), suggesting an M-2 species (only for the pseudo-molecular ion) compatible with the proposed B-type cyclic tetramer (B-type CTP). Additional evidence supporting our hypothesis is that 7-A and 7-B (despite being isobaric species) showed different relative abundances of fragments with respect to their pseudo-molecular ion: for 7-A the second most intense fragment (m/z 289.07) is about 30% of the pseudo-molecular ion relative abundance, whereas for 7-B (m/z 533.10) the most intense fragment is higher than 75%. A similar difference is noted between all the cyclic and linear oligomers in Figures 2 and 7. Hence, it is probable that the linear analogs (A- and B-type alike) can be fragmented more easily than the cyclic analogs, at least under these conditions.
Hydrogen/deuterium exchange provided a direct method to support the structural resolution of unconventional PAC in red wine and cranberry extract. With this technique, it was demonstrated that the main, most abundant PAC tetramer (referring to m/z 1153.2608, eluting at 3.9 min) in wine belonged to the proposed novel class of cyclic oligomeric B-type PAC. Moreover, this compound was identical (same retention times, exact masses, and MS/MS fragmentations) to the major tetrameric PAC found in cranberries. Traces of A-type LTP were, however, observed in cranberries and (much less) in wine at later retention times, but they were negligible with respect to the B-type CTP in both samples. In addition, a novel B-type cyclic hexameric proanthocyanidin (CHP) was also tentatively identified for the first time in wine and was assigned by analogy with the previously discovered cyclic oligomers. In fact, the identification of CHP was proposed: (1) on the basis of its exact mass; (2) on the basis of its polarity (hence its retention time) with respect to the linear analogs; (3) on the basis of the presence of only one isomer (similarly to what was observed with CTP and CPP) versus the many isomers of the linear form; (4) on the basis of its MS/MS fragmentation pattern, similar to that observed with CTP and CPP and showing fragments typical for B-type PAC.
The authors thank Kellerei Bozen (Bolzano, Italy) for providing the samples of Lagrein wine used for the analysis. The authors thank the Province of Bolzano (Italy) (Landesregierung mittels Beschluss Nr. 1472, 07.10.2013) for their financial support.
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