Phenolic Profile of Grape Canes: Novel Compounds Identified by LC-ESI-LTQ-Orbitrap-MS

Grape canes (Vitis vinifera L.) are a viticulture industry by-product with an important content of secondary metabolites, mainly polyphenols with a broad spectrum of demonstrated health benefits. Grape canes, therefore, have considerable economic potential as a source of high-value phytochemicals. In this work, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole-Orbitrap mass spectrometry (LC–LTQ-Orbitrap) was used for the comprehensive identification of polyphenolic compounds in grape canes. Identification of polyphenols was performed by comparing their retention times, accurate mass measured, and mass fragmentation patterns with those of reference substances or available data in the literature. A total of 75 compounds were identified, including phenolic acids, flavanols, flavonols, flavanonols, flavanones, and stilbenoids. The most abundant polyphenols were proanthocyanidins and stilbenoids and their oligomers. Moreover, the high-resolution mass spectrometry analysis revealed the occurrence of 17 polyphenols never described before in grape canes, thereby providing a more complete polyphenolic profile of this potentially valuable by-product.


Introduction
The bark of woody plants considered a by-product of the forestry agricultural, and wood industry. This can be an abundant source of polyphenolics compounds with high recovery yields [1][2][3]. Bark polyphenols might be esterified and used for the design of thermoplastic blends [4,5] and the developing of adhesive resins [6]. However, the most extended application of bark polyphenols compounds is its biological effects, such as antioxidant, anti-inflammatory, anti-tumor, antidiabetic, antimutagen, etc. [1,7]. In particular, grape canes, known also as vine-shoots, are one of the most important by-products in viticulture, alongside grape seeds, pomace, stalks, and skins, all of which could provide low-cost raw material for the production of high-value phytochemicals because of their rich polyphenolic content.
Polyphenolic compounds, the most important class of secondary metabolites in V. vinifera L., are synthesized by the phenylpropanoid pathway in response to biotic and abiotic stimuli [8]. The most abundant polyphenols in grape canes are oligostilbenoids and proanthocyanidins [9,10]. Stilbenoids are members of the non-flavonoid phenolic family and play an important role in the defense mechanism of plants. The concentration of stilbenoids in grape canes strongly depends on their storage/treatment after pruning [11]. Proanthocyanidins are oligomers and polymers formed by flavan-3-ol units with multiple possible linkages and different degrees of polymerization [10].
Stilbenoids, mainly (E)-resveratrol, have a wide range of health benefits, with positive effects on cardiovascular and cognitive diseases, cancer, type 2 diabetes, oxidative stress, and inflammation states [12]. Proanthocyanidins exhibit a broad spectrum of pharmacological and therapeutic benefits, including prevention of oxidative stress and degenerative diseases, gastrointestinal distress, neurological disorders, pancreatitis and various stages of neoplastic processes and carcinogenesis [13]. Besides stilbenoids and proanthocyanidins, grape canes contain other polyphenolic compounds with high biological value but in lower concentrations. The unique combination of grape phenolic compounds makes grape, raisins and grape canes, a promising source for the development of novel nutraceutical products [14].
The aim of the present work was to provide an accurate and comprehensive identification of polyphenols in grape canes using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole-Orbitrap mass spectrometry (LC-LTQ-Orbitrap) analysis, with special focus on previously unreported compounds. The novelty of this study is to extend the knowledge about polyphenols identity of grape canes for the development of additives in food, cosmetics, biomaterials, and other biobased products. Table 1 shows the 75 polyphenolic compounds identified in grape canes through LC-LTQ-Orbitrap experiments, along with their retention times (min), accurate mass, ion molecular formula (IMF), error (ppm), and the MS 2 ions used for identification. The main polyphenolic classes identified were: hydroxybenzoic acids (14), hydroxycinnamic acids (2), flavanols (mainly proanthocyanidins) (31), flavonols (3), flavanonols (3), flavanones (3), and stilbenoids (19). To the best of our knowledge, 17 polyphenols were identified for the first time in grape canes, although some of them have been previously identified in other wine by-products, such as grape seeds [31][32][33], stalks [34], pomace [27,35] and skins [31,33,36]. Figure 1 shows a base peak chromatogram of a grape cane extract.

Phenolic Acids
Phenolic acids, abundant in agro-industrial by-products [37], are of interest for their biological activity as anti-inflammatory, hepatoprotective, antioxidant, antimicrobial, cardioprotective, antidiabetic, anticancer, and neuroprotective agents [38]. Phenolic acids identified in grape cane extract can be subdivided into hydroxybenzoic and hydroxycinnamic acids and their derivatives.   Table 1. Figure 1. Base peak chromatogram of grape cane. Peaks and compounds are shown in Table 1.
Interestingly, two hydroxybenzoic acid derivatives were also identified. Gallic acid ethyl ester (m/z 197.0454, peak 12) showed an ion at m/z 169 arising from the loss of an ethyl unit (−28 Da). Gallic acid ethyl ester or ethyl gallate have been previously identified in wine extracts [41], but not in grape canes. Ellagic acid pentoside (m/z 433.0416, peak 13) was also identified and confirmed by MS 2 experiments. In the MS 2 spectrum of m/z 433, the ion at m/z 301 was due to the loss of a pentosyl unit (−132 Da) [42]. As far as we know, this is the first time that ellagic acid pentoside has been identified in grape canes.

Hydroxycinnamic Acids Derivatives
Hydroxycinnamic acids are important polyphenol precursors biosynthesized in plants from the amino acids phenylalanine and tyrosine in the shikimate pathway [43]. Hydroxycinnamic acids and their derivatives exhibit antioxidant, anti-inflammatory, antimicrobial, and ultraviolet protective effects, suggesting a potential application in anti-aging and anti-inflammatory products [44].
Two hydroxycinnamic acids were identified: (i) caftaric acid (m/z 311.0406, peak 15), with ions at m/z 179 (caffeic acid) and 149 (tartaric acid) due to the loss of a tartaric acid moiety (−132 Da) and the presence of a tartaric acid molecule, respectively [45]; and (ii) coutaric acid (m/z 295.0456, peak 16), with an ion at m/z 163 attributed to a coumaric acid molecule observed after the loss of tartaric acid (−132 Da) [31,45]. Caftaric and coutaric acids have been previously identified and quantified in wine and vine shoot extracts [22,23].

Prodelphinidins and Gallate Derivatives
Prodelphinidins have been previously identified in grape canes using a two-dimensional liquid chromatography-based method [10], but the use of a high-resolution mass analyzer, such as the LTQ Orbitrap MS, could be used by improves their characterization.
Epicatechin gallate (m/z 441.0825, peak 45) was confirmed by comparison with the standard. The MS 2 spectrum of epicatechin gallate produced two fragment ions arising from the cleavage of the ester bond: at m/z 289 for deprotonated epicatechin and at m/z 169 for a deprotonated gallic acid

Prodelphinidins and Gallate Derivatives
Prodelphinidins have been previously identified in grape canes using a two-dimensional liquid chromatography-based method [10], but the use of a high-resolution mass analyzer, such as the LTQ Orbitrap MS, could be used by improves their characterization.
Epicatechin gallate (m/z 441.0825, peak 45) was confirmed by comparison with the standard. The MS 2 spectrum of epicatechin gallate produced two fragment ions arising from the cleavage of the ester bond: at m/z 289 for deprotonated epicatechin and at m/z 169 for a deprotonated gallic acid moiety [25]. Epicatechin gallate has been previously identified in vine shoots of the Airén and Cencibel varieties [22].
Three monogallate procyanidin dimers (m/z 729.1459, peak 39; m/z 729.1449, peak 42; m/z 729.1441, peak 46) were also identified. The peaks 39 and 46 were tentatively assigned as (epi)catechin gallate (ECG)→(epi)catechin, and produced ions at m/z 603, 577, 439, 425, 407, and 289. The ion at m/z 603 corresponded to the loss of the A ring (1,3,5-trihydroxybenzene) (−126 Da) of the upper elemental unit via RDA fission [51]. The ions at m/z 577, 425, 407 and 289 showed the same fragmentation pattern as described for procyanidin dimers. The ion at m/z 439 was due to QM fission and was crucial in assigning the gallic acid ester in the upper position [51,52]. In addition, the compound at peak 42 was tentatively identified as (epi)catechin→(epi)catechin gallate, with ions at m/z 603, 577, 451, 441, 407, and 289. The main difference with peaks 39 and 46 was the presence of an ion at m/z 441 due to QM cleavage. This fragment unambiguously confirms that the gallic acid ester is at the bottom position [51]. Procyanidin dimer monogallates (m/z 729) were detected in grape canes in previous studies [10,17], although here their positions are proposed for the first time.
Epigallocatechin and gallocatechin (m/z 305.0665, peak 21; m/z 305.0662, peak 27) were tentatively identified. Fragmentation of both compounds produced ions at m/z 261, 221, 219, and 179. The ion at m/z 261 was due to loss of CO 2 (−44 Da). The ions at m/z 221, 219, and 179 arose from cleavage of the A ring and loss of −126 Da by HRF [53]. To the best of our knowledge, this is the first time that epigallocatechin or gallocatechin have been identified in grape canes.
A prodelphinidin dimer formed with units of (epi)catechin and (epi)gallocatechin (EGC) gallate (m/z 745.1400, peak 35) was tentatively assigned as (epi)catechin→(epi)gallocatechin gallate (EGCG). MS 2 of m/z 745 produced ions at m/z 593, 575, 457, 441, 423, and 305. The ion at m/z 593 was generated by the loss of a galloyl moiety (−152 Da), and at m/z 575 by the loss of gallic acid (−170 Da). The ion at m/z 457 resulted from QM cleavage and suggested a linkage between (epi)catechin and (epi)gallocatechin gallate [54]. Furthermore, the ion at m/z 305 generated by QM fission suggests that (epi)catechin and (epi)gallocatechin are positioned at the top and bottom, respectively [55]. The ion at m/z 593 underwent further fragmentation, producing an ion at m/z 441 due to RDA (−152 Da) cleavage [55]. The high-intensity ion at m/z 423 arose from the loss of a water molecule (−18 Da) from the ion at m/z 441. As far as we know, this is the first time that (epi)catechin-(epi)gallocatechin gallate has been identified in grape canes.
(Epi)gallocatechin gallate (EGCG) (m/z 457.0770, peak 38) was also tentatively identified. MS 2 of m/z 457 produced ions at m/z 331, 305, and 169. The ions at m/z 305 and 169 were formed by (epi)gallocatechin and gallic acid deprotonated units, respectively [53]. The ion at m/z 331 was generated by the HRF (−126 Da) mechanism, characteristic of flavan 3-ol monomers [56]. (Epi)gallocatechin gallate is the predominant polyphenol in green tea, and is largely responsible for the biological activity of this beverage [57]. Widely studied for its antioxidant [58], anticarcinogenic [59], and neuroprotective properties [60], (epi)gallocatechin gallate has been identified and quantified in grape seeds, including of the Pinot Noir variety [61], although to our knowledge, this has not been previously reported in grape canes.
Theaflavin (m/z 563.1191, peak 47) was likewise tentatively identified. MS 2 of m/z 563 produced ions at m/z 545, 519, 425, 407, 397, and 379. The ions at m/z 545 and 519 arose from a loss of H 2 O (−18 Da) and CO 2 (−44 Da), respectively [53]. The ion at m/z 425 was due to an RDA rearrangement of the m/z 563 precursor ion with the loss of a neutral molecule (−138 Da). Fragmentation of the ion at m/z 425 led to ions at m/z 407, 397, and 379, corresponding to losses of H 2 O (−18 Da), CO (−28 Da), and H 2 O and CO (−46 Da), respectively [53]. Theaflavins can be produced from green tea catechins (EC, ECG, EGC, and EGCG) through oxidation by polyphenol oxidase and peroxidase enzymes. This process occurs in fresh green tea leaves during the production of black tea leaves or the green tea fermentation stage [62]. Accordingly, the presence of theaflavin in grape canes was tentatively attributed to the extraction process, which provoke its formation from other flavan-3-ols. To the best of our knowledge, this is the first report of theaflavin in grape canes.
Four prodelphinidin dimers consisting of (epi)gallocatechin-(epi)catechin (m/z 593.1305, peak 20; m/z 593.1301, peak 22; m/z 593.1296, peak 24; m/z 593.1290, peak 34) were also tentatively identified. Three (epi)gallocatechin→(epi)catechins were observed at peaks 20, 24, and 34. The MS 2 spectrum of this sequence produced ions at m/z 467, 425, 407, 303, and 289. The ion at m/z 467 was due to fragmentation by HRF [M − H − 126] − on the upper unit. The ion at m/z 425 arose from RDA cleavage on the extension unit of the dimer [63], and at m/z 407 from water loss at m/z 425. These dimers were identified as (epi)gallocatechin→(epi)catechin based on the specific ions at m/z 303 and 289 derived from QM cleavage [52,63]. Peak 22 of the (epi)catechin→(epi)gallocatechin sequence showed distinctive ions at m/z 441, 423, 305, and 287. The ions at m/z 441 and 423 were generated by the RDA mechanism and the subsequent loss of a water molecule [64]. The ions at m/z 305 and 287 resulted from QM cleavage and were specific to the (epi)catechin→(epi)gallocatechin sequence [52]. Prodelphinidin dimers (m/z 593) have been detected in grape cane extracts in previous studies [10,17], although their sequences are proposed here for the first time.
Two prodelphinidin dimers consisting of (epi)gallocatechin→ ( The ion at m/z 441 can be attributed to RDA-type fragmentation and at m/z 423 to water elimination from m/z 441. The ion at m/z 305 was produced by QM cleavage between the C and D rings [64]. Prodelphinidin dimers made up of two (epi)gallocatechin units have been previously identified in red wine [52,64], although not in grape canes.
Two prodelphinidin trimers (m/z 897.1869, peak 23; m/z 897.1868, peak 25) were tentatively identified. A trimer with the sequence (epi)gallocatechin→(epi)catechin→(epi)gallocatechin was detected at peak 23, with ions at m/z 771, 729, 711, 593, and 305. The ion at m/z 771 was generated by HRF [M − H − 126] − of the C ring. The ion at m/z 729 was produced by an RDA-type mechanism on the upper unit, and the consequent loss of a water molecule led to m/z 711. The ions at m/z 593 and 305 were due to QM cleavage between the C and D rings [64] (Figure 4A), and were specific to the proposed sequence. Peak 25 corresponds to (epi)gallocatechin→(epi)gallocatechin→(epi)catechin, with ions at m/z 771, 729, 711, 593, 303, and 289. Specific ions at m/z 303 and 289 were detected, thereby indicating a QM cleavage of the interflavan bonds and formation of monomeric units, (epi)gallocatechin (-3H) and (epi)catechin, respectively ( Figure 4B) [65]. A prodelphinidin trimer (m/z 897) was previously identified in a grape cane extract [10]. The sequences of the two prodelphinidin trimers identified here are proposed for the first time.

Flavonols and Derivatives
Flavonols are biologically valuable phytochemicals associated with antioxidant and anticancer activities. In particular, myricetin has shown a wide spectrum of biological properties, including antioxidant, anticancer, anti-inflammatory, and possibly even protection against Parkinson's and Alzheimer's disease [66]. Quercetin, on the other hand, has attracted attention for its potential effects against cardiovascular diseases [67]. Three conjugated flavonols were identified in the chromatograms of grape cane extracts.
Myricetin-O-hexoside (m/z 479.0821, peak 48) showed ions at m/z 317 and 316 corresponding to the loss of a hexoside moiety (−162 Da) with concomitant H rearrangement, as usually occurs with polyphenol O-glycosides [68]. Myricetin-O-hexoside was tentatively identified by comparison with the mass spectra of previous studies using the LTQ-Orbitrap to analyze red wine [25], persimmon leaves [28], and grape pomace [27]. Although myricetin has been previously identified and quantified in vine shoot extracts [39], to our knowledge, this is the first identification of its hexoside derivatives in grape canes.
Quercetin-O-glucoside (m/z 463.0876, peak 49) was unambiguously determined and confirmed by comparison with its pure standard. The MS 2 spectrum of m/z 463 produced ions at m/z 301 and 299 due to the loss of a hexoside moiety (−162 Da) and concomitant H rearrangement, respectively. Quercetin-O-hexoside was detected previously in grape canes using a QTrap3200 LC/MS/MS system [17].
Quercetin-3-O-glucuronide (m/z 477.0674, peak 50) was also identified. The MS 2 spectrum of m/z 477 revealed an ion at m/z 301 arising from the loss of a glucuronide moiety (−176 Da). Quercetin-3-O-glucuronide has been previously identified in grape canes infected by Bois noir, a serious grapevine yellows disease [19].

Flavanonols and Derivatives
Flavanonols, also known as dihydroflavonols, are a polyphenol subclass inversely associated with diabetes in animal and in vitro models [69]. Furthermore, a high intake of dihydroflavonols has been linked with a reduced risk of diabetes in elderly persons at high risk of cardiovascular disease [70]. Thus, flavanonols, particularly dihydroquercetin (or taxifolin), have high potential value for the development of new natural drugs for the control of type 2 diabetes.
Three flavanonols were identified in the grape cane extract. Taxifolin (m/z 303.0505, peak 51) showed ions at m/z 285, 177, and 125. The ion at m/z 285 was due to the loss of a water molecule (−18 Da), whereas at m/z 177 and 125 the ions correspond to cleavage of the C ring attributed to 1,4 B − − 2H, and 1,4 A − + 2H scissions, respectively ( Figure 5) [71]. This is the first report of taxifolin in extracts from grape canes.
Molecules 2019, 24, x; doi: FOR www.mdpi.com/journal/molecules with diabetes in animal and in vitro models [69]. Furthermore, a high intake of dihydroflavonols has 214 been linked with a reduced risk of diabetes in elderly persons at high risk of cardiovascular disease 215 [70]. Thus, flavanonols, particularly dihydroquercetin (or taxifolin), have high potential value for the

Flavanones and Derivatives
Eriodictyol and its glycoside derivatives are the main flavanones found in grape canes. Eriodictyol protects against oxidative stress and could have potential application in nutraceuticals for the prevention of cardiovascular disease [73]. Eriodictyol and two of its glycoside conjugates were identified in the grape cane extract.
The MS 2 spectrum of eriodictyol-O-hexoside (m/z 449.1090, peak 54; m/z 449.1087, peak 55) produced an ion at m/z 287 generated by the loss of a hexosyl moiety (−162 Da). This is the first time that eriodictyol-O-hexoside is reported from grape canes.

230
Eriodictyol and its glycoside derivatives are the main flavanones found in grape canes.

231
Eriodictyol protects against oxidative stress and could have potential application in nutraceuticals 232 for the prevention of cardiovascular disease [73]. Eriodictyol and two of its glycoside conjugates were 233 identified in the grape cane extract.

234
The

244
The most relevant stilbene is resveratrol, associated with activity against cardiovascular diseases, 245 neurodegenerative diseases, and cancer [76]. In grape canes, the most abundant polyphenolic 246 Figure 6. Proposed formation of product ions at m/z 135 and 151 for eriodictyol.

Polyphenol Extraction from Grape Canes
Grape canes were handled in a room with light filters to prevent photodegradation and oxidation of the polyphenols. The extraction was done following a previously reported procedure with minor modifications [82].
Grape canes (0.5 g, n = 3) were homogenized and vortexed for 1 min with 4 mL ethanol/water (80:20, v/v) and then sonicated in an ultrasound bath (Bandelin electronic GmbH&Co.KG, Berlin, Germany) for 10 min. The grape cane extract was centrifuged at 4000 RPM for 5 min at 4 • C. The supernatant was collected and the extraction procedure was repeated twice. The supernatants were combined and evaporated under nitrogen flow, and the residue was reconstituted into 0.1% of aqueous formic acid (5 mL). The extract was filtered by 0.20 µm PTFE (Waters Corporation, Mildfore, MA, USA) into an amber vial. Samples were stored at −20 • C until analysis by LC LTQ-Orbitrap.
The LC system was coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Hemel Hempstead, UK) used for accurate mass measurements and equipped with an ESI source operated in negative mode. Operation parameters were as follows: source voltage, 4 kV; sheath gas, 20 a.u. (arbitrary units); auxiliary gas, 10 a.u.; sweep gas, 2 a.u.; and capillary temperature, 275 • C. Default values were used for most other acquisition parameters (FT Automatic gain control (AGC) target 5 × 10 5 for MS mode and 5·× 10 4 for MS n mode). Grape cane samples were analyzed in full scan mode at a resolving power of 30,000 (FWHM at m/z 400) and data-dependent MS/MS events acquired at a resolving power of 15,000. The most intense ions detected in the full scan spectrum were selected for the data-dependent scan. Parent ions were fragmented by high-energy C-trap dissociation (HCD) with normalized collision energy of 35 V and an activation time of 10 ms. The mass range in Fourier transformation mass spectrometry (FTMS) mode was from m/z 100 to 1000 [28]. Instrument control and data acquisition were performed with Xcalibur 3.0 software (Thermo Fisher Scientific).

Conclusions
The use of LC-LTQ-Orbitrap-MS allowed a comprehensive profiling of polyphenols in a grape cane extract. The characterization was carried out based on accurate mass measurement with low error (<3.1 ppm) and MS 2 spectrum data. The polyphenolic compounds were confirmed by comparisons with pure standards whenever possible, as well as by referring to the literature. A total of 75 polyphenolic compounds were identified or tentatively characterized, 17 of them reported for the first time in grape canes. Most of the identified polyphenols were hexoside derivatives, such as syringic acid hexoside, hydroxybenzoyl hexoside, ellagic acid hexoside, myricetin-O-hexoside, eriodictyol-O-hexoside, and resveratrol dimer-O-hexoside. Additionally, an exhaustive analysis of proanthocyanidins showed for the first time the presence of pentameric procyanidins and (epi)gallocatechins, and the specific sequence of each prodelphinidin compound.
The reported results broaden knowledge of the polyphenol profile of grape canes and may be useful for further investigations related to the production of high-added-value food additives based on this by-product.