Elucidating the Structure of Oligomeric Cyclic Procyanidins

Isotopic H/D Exchange in Red Wine and Cranberry Extracts

  • © S.ta Magdalener by Bolzano, Italy© S.ta Magdalener by Bolzano, Italy
  • © S.ta Magdalener by Bolzano, Italy
  • Tab. 1 Retention times for the species of interest (HPLC-HRMS analysis in water)
  • Fig. 1 (A) Non-cyclic tetrameric PC with one A-linkage (A-LTP). (B) Cyclic (crown) tetramer (B-CTP). (C) B-type non-cyclic tetrameric PC (B-LTP). (D) Deuterated non-cyclic tetrameric PC with one A-linkage (A-LTP-d19). (E) Deuterated cyclic (crown) tetramer (B-CTP-d20). (F) Deuterated B-type non-cyclic tetrameric PC (B-LTP-d20). Inter-(epi)catechin bonds and stereogenic centers’ configurations were arbitrarily indicated and must be considered unresolved in this work.
  • Fig. 2: For each layer from A to F, the comparison between wine (top) and cranberry extract (CE) (down) is reported. A) Extracted Ion Chromatogram (EIC, H2O) of m/z 1153.2608 CTP; B) Full MS (ESI+, m/z 1153-1160 range shown) at 3.8±0.1 min in water; C) EIC (ESI+, D2O) of m/z 1174.3926 CTP-d20; D) Full MS (ESI+, m/z 1174-1180 range shown) at 5.0 min in D2O; E) MS/MS spectrum in H2O at 3.8±0.1 min of m/z 1153.26 species; F) MS/MS spectrum in D2O at 5.0±0.1 min of m/z 1174.39 species.

Cyclic oligomeric (crown) procyanidins have been recently proposed in wine. Hydrogen/deuterium exchange (HDX) allowed to unambiguously confirm their presence. Crown procyanidins were identified in a cranberries extract, too. The identification in red wine of a cyclic (crown) hexamer (CHP) was also proposed.


Procyanidins (PC) are natural compounds composed of (+)-catechin and (-)-epicatechins [1,2]. A novel class of crown procyanidins has been also proposed [3]. These are characterized by head/tail linkages that make them cyclic. However, a structural ambiguity was identified [4]: they share their elemental composition with previously known PC with one A-linkage (A-type PC). A-type PC were reported in cranberries, grape and other fruits [5]. Hydrogen/deuterium exchange (HDX) offered a valuable instrument for their structural resolution [6]. HDX successfully confirmed the structure of the novel crown PC in wine and cranberries.



All material was purchased from Sigma Aldrich (Milano, Italy). Solvents and salts were MS grade. D2O was 99.9%D. Wine (Lagrein, 2016 vintage) was donated by a local winery (Kellerei Bozen, Bolzano). Cranberries were purchased from a local market.

Sample preparation

A volume of 135 mL of acetone/water/acetic acid (70:29.5:0.5 v/v) was added to 15 g of dried cranberries [5]. The mixture was blended and sonicated in a water bath at 99 W for 5 min (50oC). Then, it was vortexed and centrifuged (3300 rpm). The cranberries extract and a red wine (not extracted) were concentrated at 9–10 mbar followed by 30 min of gentle N2 flux and reconstituted to a volume 10 to 30 times smaller than the initial one. The samples were filtered (0.2 μm, regenerate cellulose). For HDX, the samples were first dried and then reconstituted for 3/4 times in neat D2O (5% CH3CN).


The method was adapted from a previous report [4,7]. A Q Exactive HRMS analyser (Thermo Fisher Scientific, Rodano, Milano, Italy) was connected in parallel to a 1260 Agilent HPLC with a 16-channel diode array detector (Agilent Technologies, Cernusco sul Naviglio, Milano, Italy).

The separation was run on a ODS Hypersyl C18 LC column (125 mm × 4.6 mm, 5 μm, Thermo Fisher Scientific) protected with a guard column (ODS Hypersil, 5 μm pore size,10 × 4 mm drop-in guards, Thermo Fisher Scientific). The mobile phases were A (0.1%v/v formic acid/0.02 mol·L-1 ammonium formate in water or 0.1% v/v formic acid-d2/0.02 mol·L-1 ammonium formate-d5 in D2O) and phase B (0.1%v/v formic acid/ammonium formate(sat.) in CH3CN or 0.1% v/v formic acid-d2/ammonium formate-d5 (sat.) in CH3CN). The separation was carried out at 1 mL·min-1 with the following program: 5-25 % B, 0-21 min; 95% B 22-27 min; 5% B 28-32 min.

ESI(+) with a mass range of m/z 500-2000 was performed at +3.00 kV. Lock masses were employed throughout the experiments. Data-dependent LC-MS/MS experiments were run on concentrated samples at a normalized collision energy (NCE) of 15. MS results were collected and analysed by Xcalibur 3.1 (Thermo Fisher Scientific). MS/MS fragments interpretation was performed on the basis of comparison with the cited literature, and aided by Mass Frontier 7.0 (Thermo Fisher Scientific) software. For HDX, the samples were first dried and then reconstituted for 3/4 times in neat D2O (5% CH3CN). For HDX the column and lines A and B were conditioned with deuterated mobile phases. Lock masses in the MS method were accordingly modified.

Results and Discussion

Table 1 summarizes the retention times for the species of interest. CHP in Tab. 1 (C90H72O36, CHP) was assigned according to its predicted structure, MS/MS fragmentation and relative polarity [4]. The main fragmentation mechanisms contributing for all PC were the retro Diels-Alder ring opening, the quinone methide monomer loss and the heterocyclic ring fission. Their discussion is reported elsewhere [4]. Overall, the crown PC fragmented less than the non-cyclic analogues.
Nonetheless, the proposed novel crown PC share elemental composition with some PC analogues of the A-type [5]. HDX can differentiate them because they differ in the number of exchangeable (phenolic) protons. In Figure 1, these difference is explained.

1a and 1b are isobaric species. However, all the hydroxy protons are exchanged under HDX. Therefore, 1d and 1e are not isobaric anymore: the m/z 1153.2608 species 1a would add 20.126 Da whereas the m/z 1153.2608 species 1b would add 21.132 Da. Also, the comparison of the MS/MS patterns for wine and cranberries in water and D2O can help, as reported in Fig. 2, showing the HPLC-HRMS/MS results. Under HDX the m/z 1153.2608 tetramer sums 21.1 Da, as predicted for CTP (EIC in Fig. 2D). Besides, MS/MS spectra in H2O at ~3.8 min and D2O at ~5 min are identical for wine and the cranberries extract (E-F). The obtained fragments in water (E) differ from previously reported fragmentation of A-type linear tetrameric procyanidin [8]. HDX showed that the main PC tetramer m/z 1153.2608 (rt=3.8 min) was the crown tetrameric PC in both wine and cranberries.


Edoardo Longo1, Fabrizio Rossetti2, Matteo Scampicchio1, Emanuele Boselli1

1Faculty of Science and Technology, Free University of Bozen-Bolzano, Bolzano, Italy
2Department of Agricultural, Food, and Environmental Sciences, Università Politecnica delle Marche, Ancona, Italy

Emanuele Boselli
Faculty of Science and Technology
Free University of Bozen-Bolzano
Bolzano, Italy


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