A Fragmentation Study on Four Oligostilbenes by Electrospray Tandem Mass Spectrometry

Abstract Oligostilbenes have attracted much interest due to their intricate structures and diverse bioactivities. In this study, two stilbene dimers, (−)-7,8-cis-ε-viniferin (1) and carasiphenol A (2), and two trimers, suffruticosol A (3) and suffruticosol C (4), were investigated by electrospray ionization ion-trap time-of-flight multistage mass spectrometry (ESI-IT-TOF-MSn). Based on the MSn study, the fragmentation pathways and diagnostic ions of four oligostilbenes in both positive and negative modes were proposed. The consecutive elimination of phenol (C6H6O) and resorcinol (C6H6O2) moieties were the particular dissociation for oligostilbenes due to the presence of 1,2-diphenylethylene nucleus. The present MSn fragmentation study will provide valuable information for the fast characterization of oligostilbenes from complicated natural mixtures. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-019-0212-3) contains supplementary material, which is available to authorized users.


Introduction
Natural stilbenes are an important group of polyphenols characterized by the presence of 1,2-diphenylethylene nucleus [1]. Naturally occurring stilbenes always have intricate structures with different numbers of stilbenes and polymeric types [2]. Stilbenes as the defensive chemicals of plants are revealed with diverse bioactivities including anti-tumor, anti-oxidant, anti-inflammatory, Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1365 9-019-0212-3) contains supplementary material, which is available to authorized users.
Mass spectrometry (MS) with high sensitivity and resolution is one of the most efficient method in analyzing natural products [8][9][10]. Tandem MS techniques have advantages in ascertaining the relationship between precursor and product ions, by which the fragmentation rules and diagnostic ions of complicated compounds can be proposed [11,12]. In this paper, we report the MS n fragmentation rules of four oligostilbenes, (−)-7,8-cis-εviniferin (1), carasiphenol A (2), suffruticosol A (3) and suffruticosol C (4), by electrospray ionization ion-trap time-of-flight (ESI-IT-TOF) mass spectrometer to provide reference for their fast characterization from natural sources.

Results and Discussion
The first-stage MS of compounds 1-4 ( Fig. 1) in both positive and negative ion modes were acquired in automatic pattern, by which their protonated ([M+H] + ) and deprotonated ([M−H] − ) molecule ions were readily detected. For compounds 1, 3 and 4, the [M+HCOO] − ions in negative mode were also obtained due to the application of formic acid in the solvent [13]. The subsequent MS n studies on compounds 1-4 in both positive and negative modes were performed, from which their fragmentation pathways were proposed (Figs. 2,3,4,5,6,7,8,9). It should be noted that alternative ways of fragmentation that can reasonably interpret the product ions are also possible in addition to the proposed pathway. For example, the negative charge can be present at any hydroxy group rather than the position denoted.

MS n Fragmentations of (−)-7,8-cis-ε-Viniferin (1) in Positive Mode
In the single-stage mass spectrum of (−)-7   The similar fragmentation was also observed for ion 1C, from which two product ions at m/z 255 (1H) and m/z 239 (1J) were obtained corresponding to neutral loss of a C 6 H 6 O or a C 6 H 6 O 2 part. When ion 1H was selected as the precursor ion to perform MS 3 experiment, two ions 1K (m/z 227) and 1M (m/z 199) were formed by the successive loss of two CO molecules. Similarly, the ion 1F (m/z 315) was well explained by the loss of a molecule of CO from 1D [15][16][17]. In addition, a MS 2 ion at m/z 215 (1L) was also observed from 1A, but its fragmentation pathway was still unclear (Fig. 2).   (Fig. 3).  (Fig. 4).

MS n Fragmentations of Suffruticosol A (3) in Positive Mode
The MS 2 study on [M+H] + ion gave rise to the fragments at m/z 587 (3B), 575 (3C), 493 (3D), 481 (3E) and 321 (3J). The production of ion 3E (m/z 481) was verified as the successive elimination of a C 7 H 6 O and a C 6 H 6 O part from 3A.
The ion 3E could further generate ion at m/z 387 (3G) and 371 (3H) by the neutral loss of a phenol (C 6 H 6 O) and a resorcinol (C 6 H 6 O 2 ) moiety [15][16][17]. With the elimination of a molecule of H 2 O, the ion at m/z 369 (3I) was obtained from 3G. Similarly, the fragment 3F was produced from 3D by the loss of a phenol (C 6 H 6 O) part (Fig. 6).

MS n Fragmentations of Suffruticosol A (3) in Negative Mode
When the [M−H] − ion was chosen for MS 2 study, the product ions at m/z 585 (3c) and 573 (3d) were generated due to the loss of C 6 H 6 O and C 7 H 6 O moieties [15][16][17]. The following MS 3 investigation on ion 3c gave rise to fragments at m/z Fig. 9 Proposed fragmentation pathways of suffruticosol C (4) in negative mode 1 3 543 (3e), 491 (3f), 479 (3g) and 475 (3h), which could be explained by the elimination of C 2 H 2 O, C 6 H 6 O, C 7 H 6 O and C 6 H 6 O 2 . The ion 3j (m/z 385) was deduced from 3g by the neutral loss of a molecular of phenol (C 6 H 6 O) moiety. Two MS 3 fragments at m/z 357 (3k) and 341 (3l) were obtained from 3i, which were well in accordance with the departure of a phenol (C 6 H 6 O) and a resorcinol (C 6 H 6 O 2 ) parts (Fig. 7).

MS n Fragmentations of Suffruticosol C (4) in Positive Mode
The  [15][16][17]. The ion at m/z 453 (4F) was deduced from 4E by the elimination of a molecule of CO, and further gave rise to 4I (m/z 359) and 4J (m/z 265) which was well explained by the consecutive loss of two phenol (C 6 H 6 O) moieties. The ion at m/z 371 (4H) was generated from 4E by the departure of a C 6 H 6 O 2 moiety (Fig. 8).

Apparatus and Analytical Conditions
All of the MS n experiments were performed on the LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan). Accurate masses were calibrated using sodium trifluoroacetate (CF 3 CO 2 Na) clusters. MS experiments were performed in automatic pattern, and MS n experiments were achieved in direct mode. The MS parameters are in accordance with the previous report [18].

Chemicals and Samples
Acetonitrile (CH 3 CN) of HPLC grade was purchased from Merck Co., Ltd., Germany, and formic acid was bought from Aladdin Chemistry Co., Ltd., China. Deionized water was purified using a MingChe™-D 24UV Merck Millipore system (Merck Millipore, Shanghai, China). Compounds 1-4 were isolated from the seeds of Paeonia lactiflora Pall. in our previous investigation. Samples were diluted in MeOH at the concentration of 0.5 mg/mL.

Conclusion
The ESI multi-stage mass spectra (MS n ) of four oligostilbenes were studied for the first time by LCMS-IT-TOF, by which their fragmentation pathways were deduced. The consecutive elimination of phenol (C 6 H 6 O) and resorcinol (C 6 H 6 O 2 ) moieties from the precursor ions was the particular dissociation due to the presence of 1,2-diphenylethylene nucleus in the structure. Interestingly, the elimination of a C 7 H 6 O moiety was always detected due to the fracture of the double bond in 1,2-diphenylethylene nucleus, and this fragmentation pathway might be impelled by the rearrangement of the free radical into a stable conjugated system (e.g. tropone). Based on the fragmentation rules deduced above, (−)-7,8-cis-ε-viniferin (1), carasiphenol A (2), suffruticosol A (3) and suffruticosol C (4) could be well differentiated by their respective ion pars of 455-215, 429-267, 681-321 and 681-359 in positive mode, and 453-359, 427-307, 679-451 and 679-447 in negative mode. The present MS n fragmentation study will provide valuable information for the fast characterization of oligostilbenes from complicated natural mixtures.