*3.4. Phenolic Profile of the IC (GCPPE+HP-β-CD+MD) by LC-ESI-LTQ-Orbitrap-MS*

We performed a targeted analysis of phenolic compounds in the IC (GCPPE+HP-*β*-CD+MD) using LC-ESI-LTQ-Orbitrap-MS. Table 2 shows 42 identified compounds with their accurate mass, theoretical mass, retention times (min), molecular formula, error (ppm) between the found mass and the accurate mass of each compound, and the MS/MS fragment ions with their respective intensities used for identification. The identification was further supported by comparisons with mass spectra databases and the literature. In addition, 13 phenolic compounds were identified by comparing the retention times and their masses with pure standards. We have reported the fragmentation patterns of most of these compounds in previous studies using an analytical [8] and pilot-scale extraction [2].

*Antioxidants* **2021**, *10*, 1130

**Table 2.** Identification of phenolic compounds in the IC (GCPPE+HP-*β*-CD+MD), an adduct of *β*-CD and a complex between *β*-CD with (*epi*)catechin using LC-ESI-LTQ-Orbitrap-MS in negativemode.


153

*Antioxidants* **2021**, *10*, 1130


**Table2.***Cont.*

Of the 42 compounds identified in the present work, 22 had been previously detected in both the analytical and pilot-plant extractions [2,8]: gallic acid (*m*/*z* 169.0138, −2.41 ppm), monogalloyl-glucose (*m*/*z* 331.0662, −2.65 ppm), protocatechuic acid-*O*-hexoside (1) (*m*/*z* 315.0718, −1.07 ppm), protocatechuic acid (*m*/*z* 153.0190, −2.16 ppm), protocatechuic acid-*O*-hexoside (2) (*m*/*z* 315.0717, −1.30 ppm), syringic acid hexoside (*m*/*z* 359.0933, 2.21 ppm), catechin (*m*/*z* 289.0722, 1.43 ppm), epicatechin (*m*/*z* 289.0715, −1.00 ppm), restrytisol (A or B) (*m*/*z* 471.1441, −1.86 ppm), taxifolin (*m*/*z* 303.0508, −0.78 ppm), astilbin (*m*/*z* 449.1084, −1.12 ppm), stilbenoid heterodimer (caraphenol B/C) (*m*/*z* 469.1292, −0.06 ppm), eriodictyol-*O*-glucoside (*m*/*z* 449.1089, −0.04 ppm), stilbenoid dimer (*m*/*z* 469.1292, −0.07 ppm), pallidol (*m*/*z* 453.1349, 2.24 ppm), (*E*)-resveratrol (*m*/*z* 227.0715, 0.77 ppm), stilbenoid dimer (resveratrol dimer) (*m*/*z* 453.1351, 1.57 ppm), resvertarol-*O*hexoside (*m*/*z* 615.1868, −0.62 ppm), eriodictyol (*m*/*z* 287.0558, −0.92 ppm), hopeaphenol (*m*/*z* 905.2607, −2.24 ppm), isohopeaphenol (*m*/*z* 905.2588, 2.15 ppm) and (*E*)-ε-viniferin (*m*/*z* 453.1348, 1.03 ppm). These findings indicate that these compounds are stable through each step of the production process, including microencapsulation.

On the other hand, seven compounds identified in the microencapsulated extract had previously been detected only in the analytical extraction [8]: procyanidin dimer (1) (*m*/*z* 577.1348, −0.69 ppm), procyanidin dimer (2) (*m*/*z* 577.1346, −0.35 ppm), epicatechin gallate (*m*/*z* 441.0821, −1.30 ppm), (*E*)-piceatannol (*m*/*z* 243.0665, 0.79 ppm), viniferin diglycoside (*m*/*z* 777.2397, −0.36 ppm), (*E*)-ω-viniferin (*m*/*z* 453.1346, 0.62 ppm), and stilbenoid tetramer (vitisin A/B/C/D) (*m*/*z* 905.2599, −3.05 ppm). Thus, although these compounds were not detected in the pilot-scale extraction, they were recovered after the microencapsulation process. Similar to our results, the incorporation of *β*-CD enabled an effective and selective recovery of flavan-3-ols [41] and stilbenes [42], resulting in a cleaner analytical extract phenolic profile. Additionally, three compounds previously detected in the pilot-scale extraction [2]: protocatechuic aldehyde (*m*/*z* 137.0241, −2.02 ppm), kaempferol-3-*O*-glucoside (*m*/*z* 447.0931, −0.49 ppm), and ethyl protocatechuate (*m*/*z* 181.0504, −1.02 ppm), were also recovered and identified in the microencapsulated extract.

Finally, ten compounds were identified only in the IC (GCPPE+HP-*β*-CD+MD), and not in the analytical or pilot extracts: five flavonoids, three stilbenes, an adduct of *β*-CD, and a complex of *β*-CD with (*epi*)catechin.

*Flavonoids.* The taxifolin isomer (*m*/*z* 303.0503, −2.32 ppm) showed ions at *m/z* 285.0390, owing to the initial loss of a water molecule and ions at *m*/*z* 177.0184 and 125.0237 due to cleavage of the C ring, respectively. Quercetin (*m*/*z* 301.0354, 0.06 ppm) was identified and confirmed by comparison with a pure standard. Myricetin (*m*/*z* 317.0301, −0.63 ppm), tentatively identified by its fragmentation pattern, produced ions at *m*/*z* 178.9981 (1,2A−) and 151.0032 (1,3A−) due to retro-Diels–Alder fragmentation [43], and at *m*/*z* 192.0058 due to the loss of the B ring. Although they were not identified in our previous studies, these compounds have been recovered, detected, and quantified by other authors using microwave-assisted, subcritical water, and conventional extraction techniques [44].

Dihydrokaempferol-*O*-rhamnoside (engeletin) (*m*/*z* 433.1139, −0.38 ppm), a compound previously reported in grape stems [45], was tentatively identified and showed fragment ions at *m*/*z* 269.0446, 287.0550, and 259.0603. An undefined tetrahydroxyisoflavanone (*m*/*z* 287.0561, −0.18 ppm) gave product ions at *m*/*z* 259.0602, 243.0652, and 201.0547, and was provisionally identified as 2,6,7,4 -tetrahydroxyisoflavanone, based on the exact mass and fragmentation pattern. However, as dihydrokaempferol and eriodictyol chalcone have similar structures, the identity of this compound could not be accurately defined using our spectrometric approach.

*Stilbenes*. A stilbenoid dimer (maackin, Figure 3A) (*m*/*z* 485.1242, 0.01 ppm) showed product ions at *m*/*z* 467.1125, 375.0865, and 363.0863, which were generated by the loss of a water molecule (18 Da), resorcinol (110 Da), and 2-hydroxy-4-methylenecyclohexa-2,5-dienone (122 Da), respectively [46]. This compound has a structure consisting of two

piceatannol units, and the most likely assignment is maackin A, which was identified previously in *V. vinifera* stalks [47].

**Figure 3.** Representative stilbenes tentatively identified in the microencapsulated extract. (**A**) Maackin (C28H22O8); (**B**) Viniferol E (C56H44O13); (**C**) Viniphenol A (C84H64O18).

A stilbenoid tetramer (Figure 3B) (*m*/*z* 923.2680, 0.68 ppm) was tentatively identified as viniferol E and yielded product ions at *m*/*z* 905.2576, 881.2573, 801.2318, 783.2209 and 707.1898. The product ions at *m*/*z* 905.2576 and 881.2573 were due to a loss of H2O (18 Da) and C2H2O (42 Da), respectively, and at *m*/*z* 801.2318 probably to the loss of the group C7H6O2 (122 Da). The ion at *m*/*z* 801.2318 was further fragmented to ions at *m*/*z* 783.2209 and *m*/*z* 707.1898 by the loss of a water molecule (18 Da) and a phenol group (94 Da), respectively. Viniferol E was previously detected and quantified from grapevine canes by subcritical water extraction [48].

A stilbenoid hexamer, viniphenol A (Figure 3C) (*m*/*<sup>z</sup>* 679.1969 [M − 2H]2<sup>−</sup>, −1.12 ppm), was detected as a doubly charged ion with product ions at *m*/*z* 905.2584, 585.1543, 491.1126, 453.1333 and 359.0914. The product ion at *m*/*z* 905.2584 shows the presence of a stilbenoid tetramer molecule, probably formed by the loss of a stilbenoid dimer (454 Da) from the deprotonated hexamer. The high-intensity product ion at *m*/*z* 585.1543 could be attributed to the loss of a phenol group (94 Da) from the stilbenoid trimer (*m*/*z* 679). The product ion at *m*/*z* 585.1543 underwent fragmentation to ions at *m*/*z* 491.1126 and *m*/*z* 359.0914, which could be attributed to the loss of a phenol group (94 Da) and a stilbenoid dimer (226 Da), respectively. Finally, a low intensity stilbenoid dimer fragment was observed at *m*/*z* 453.1333. Viniphenol A was previously isolated from *V. vinifera* stalks by centrifugal partition chromatography, while its structure was proposed based on the analysis of spectroscopic data and molecular modeling under NMR conditions [47].

According to the supplier, the HP-*β*-CD used for encapsulation has a maximum *β*-CD impurity of 1.5%. The presence of [*β*-CD+ HCOO]— (*m*/*<sup>z</sup>* 1179.3679, −0.04 ppm) and a complex of (*epi*)catechin with *<sup>β</sup>*-CD [*β*-CD +(*epi*)catechin]— (*m*/*<sup>z</sup>* 1423.5708, −0.71 ppm) were detected and identified by comparison with the mass spectra reported by Zy˙ zelewicz et al. [ ˙ 49]. The detection of this complex by mass spectrometry confirmed the interaction between polyphenols and cyclodextrins, in agreement with the SEM and FTIR analysis.
