Detailed phenolic composition of Vidal grape pomace by ultrahigh-performance liquid chromatography–tandem mass spectrometry

Journal of Chromatography B 1068–1069 (2017) 201–209

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb

Detailed phenolic composition of Vidal grape pomace by ultrahighperformance liquid chromatography–tandem mass spectrometry Lanxin Luoa, Yan Cuib, Shuting Zhangc, Lingxi Lib, Hao Suoc, Baoshan Sunb,d,

MARK

⁎

a

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, 110016, Shenyang, China School of Functional Food and Wine, Shenyang Pharmaceutical University, 110016, Shenyang, China c School of Pharmacy, Shenyang Pharmaceutical University, 110016, Shenyang, China d Pólo Dois Portos, Instituto National de Investigação Agrária e Veterinária, I.P., Quinta da Almoinha, 2565-191, Dois Portos, Portugal b

A R T I C L E I N F O

A B S T R A C T

Keywords: Vidal grape Phenolic composition UPLC analysis FT-ICR mass spectrometry QqQ mass spectrometry

Vidal Blanc grape (Vitis vinifera cv.) is the predominant white grape variety used for the production of icewine in China’s Liaoning province. In this paper, the development and validation of the method by ultrahigh-performance liquid chromatography-tandem mass spectrometry has been performed for determination of the detailed phenolic composition in the skin, seed and stem of Vidal grapes. The validation of the method was realized by calculating the linearity, repeatability, precision, stability and the limits of detection (LOD) and quantification (LOQ) of standard solutions. All the curves exhibited good linearity (r2 > 0.9997) and the LOD and LOQ were in the range of 0.002–0.025 and 0.006–0.086 μg/ml, respectively. Good repeatability (RSD < 4.3%) and stability (RSD < 3.7%) were also found. Results confirmed that the developed method was more effective and sensitive for simultaneous determination of the major phenolic compounds in Vidal grape pomace. The optimized and validated method of ultrahigh-performance liquid chromatography tandem two complementary techniques, fourier transform ion cyclotron resonance mass spectrometry and triple-quadrupole mass spectrometry, allowed to identify and quantify up to 35 phenolic compounds in Vidal grape pomace, which has, as far as we know, been reported this grapevine variety for the first time. Seeds, skins and stems exhibited different qualitative and quantitative phenolic profiles. These results provided useful information for recovery of phenolic antioxidants from different parts of icewine pomace.

1. Introduction Plants have developed sophisticated processes for example the production of various of secondary metabolites for enhancing their survival [1]. These secondary metabolites which are classified into three major groups i.e., terpenes, alkaloids and phenolic compounds, based on their synthesis from different metabolic pathways have diverse biological activities for human health, including uses as medicinal compounds and serve for various purpose, for instance providing resistance to the plants against herbivores and pathogens, and in protection against ultraviolet light [2]. Phenolic compounds as the one group of secondary metabolites are the most important quality parameters of grapes and wines [3]. These compounds are not only responsible for the sensory properties such as color, astringency and bitterness of wine [4], but also contribute to its health benefits because of their various biological activities like antioxidant, anti-inflammatory and anti-cancer activities [5–7]. In grape clusters, phenolic compounds present mainly in the solid parts (skin,

⁎

seed and stem) and show a great diversity of structures, ranging from simple molecules (monomers and oligomers) to polymers. Furthermore, the pomace from white and red winemaking presents the distinguished phenolic profiles. The pomace from white winemaking does not suffer maceration process and thus preserves nearly all polyphenols in grape cluster, while that from red winemaking losses important part of polyphenols during alcoholic fermentation/maceration process [8]. Therefore, grape pomace from white winemaking is more interesting and useful as rich sources of polyphenols for wine industry. Vidal Blanc (Vitis vinifera cv.) is one of the predominant white grape varieties, which is used for the production of icewine because it has large cylindrical clusters, with medium-sized, thick-skinned berries which are diseases resistant [9]. Vidal grape’s key viticultural feature is that it possesses the requisite physiological properties to withstand the rigours of the climate [10]. For ice wine production, Vidal grapes can be frozen naturally on the vine and be pressed when still frozen, leading to a small amount of dessert wine and highly concentrated in sugar and

Corresponding author at: School of Functional Food and Wine, Shenyang Pharmaceutical University, 110016, Shenyang, China. E-mail addresses: [email protected], [email protected] (B. Sun).

http://dx.doi.org/10.1016/j.jchromb.2017.10.031 Received 2 July 2017; Received in revised form 9 October 2017; Accepted 14 October 2017 Available online 16 October 2017 1570-0232/ © 2017 Published by Elsevier B.V.

Journal of Chromatography B 1068–1069 (2017) 201–209

L. Luo et al.

pressing in “LongYuanQuan” winery (Huanren, China). Seeds, skins and stems were manually separated from the pomace and grounded under liquid nitrogen, then stored under −80 °C until use. The reference compounds of (+)-catechin, (−)-epicatechin, epicatechin gallate, gallic acid, ferulic acid, p-coumaric acid, isoquercetin, astragalin were purchased from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Procyanidin dimers B1, B2, B3, B4 and B7 were isolated in our laboratory by HSCCC and semi-preparative HPLC as described previously [22]. Hydrochlorothiazide as the internal standard [23] was purchased from the National Institute for Control of Biological and Pharmaceutical Products (Beijing, China). Organic solvents of MS grade were purchased from Fisher Scientific (USA). Analytical grade methanol and acetone purchased from Shandong Yuwang industrial Co., Ltd. (Shandong, China) was used for sample preparation.

other flavor compounds [10,11]. Furthermore, Vidal is a high acid cultivar prone to overcropping, which enables it to have a large yield for icewine production [12]. Therefore, the yield of pomace from icewine production is also as high as 70–75% (w/w), which is much higher than the table wine (20%). Because the icewine pomaces are largely produced and discarded as waste each year by ice-wineries, there is a renewed interest in studying the detailed phenolic composition of these materials, which may be used as a cheap source for bioactive compounds obtained. Grape phenols consist of a wide range of structures diversely distributed in every part of the berry, but they are present mainly in the seeds, skins and stems. The most abundant phenolic compounds in white grape skins include procyanidins, flavonols and phenolic acid, some skins of white varieties like Albariño grapes [13] also contain resveratrol and piceid. while dimers, trimers and polymeric forms, also called procyanidins, are present mainly in grape seeds. And the major polyphenols in white grape stems are procyanidins and flavonols. It is also well known that different phenolic compounds may show different biological and antioxidant properties [14]. There is an imperative need for a precise and sensitive method to determine the phenolics in Vidal grape pomace. Traditionally, phenolic composition has been determined by high performance liquid chromatography (HPLC), coupled with ultraviolet (UV), or diode array (DAD) detectors-tandem mass spectrometry [4,15] which could cause long analysis time and low separation resolution because of the complexity in Vidal grape pomace. However, ultrahigh performance liquid chromatofraphy (UPLC) as a new trend in separation science, presents distinguished advantages over traditional HPLC instruments due to its high pressures (up to 108 Pa) and sub–2 μm particles in column, reaching to high resolution and efficiency, short analysis time and less sample loads, which are especially important for separating complex substance [16]. For mass spectrometry, fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) is an important technique to study the composition and structure of small molecules, DNA, proteins and protein/DNA assemblies [17]. Recently, it is also been used for complex compounds and metabolome analysis because of its ability to achieve an ultra-high resolving power by accumulating time-domain transient signals in different time-domain data sets with high mass accuracy [18]. FT-ICR/MS is an important method for the phenolic compounds identification in Vidal pomace for it can give a precise molecular weight and formula. Triple-quadrupole instrument (QqQ/ MS) substantially improves the selectivity and sensitivity compared to single-stage MS because of the elimination of isobaric interference and the reduction of chemical noise [19]. When QqQ/MS in full scan mode, it shows a bad signal-to-noise-ratio but MS/MS techniques such as product ion scan (PIS), precursor ion scan (PrI), and neutral loss scan (NL) allow phenolic composition to be screened [20,21]. Compared to other MS instruments, QqQ system is more stable and more sensitive and provides better quantification results, especially for low levels in complex matrices and compounds. To our best knowledge, Vidal variety grape has not been well characterized regarding to its phenolic composition. Furthermore, there are no reports on the use of UHPLC-FT-ICR/MS and UPLC-QqQ/MS for the qualitative and quantitative analysis of phenolic composition in grape pomace. Aim of this research is to identify and quantify the detailed phenolic compostion in the skins, seeds and stems of Vidal grape variety with an optimized UHPLC-FT-ICR/MS and UPLC-QqQ/MS methodology. Our results provide an useful information for selecting suitable and economical sources to prepare bioactive polyphenols.

2.2. Preparation of standard solutions All the standard solutions were freshly prepared. Each standard was accurately weighed, and then dissolved in methanol-water (50:50, v/v) to give the stock solution. Working standard solutions containing 8 reference standards were prepared by diluting the stock solutions with methanol-water (50:50, v/v) to a series of proper concentrations. To each standard solution, an aliquot of stock solution of hydrochlorothiazide (I.S.) was added to make up a final concentration of 5.94 μg/ml. 2.3. Preparation of sample solutions Total phenolic compounds were extracted from the powder of skins, seeds and stems with the method described previously [8]. Briefly, a 20 g portion of each powder was extracted using 300 ml of methanolwater (80/20; v/v) followed by 300 ml of acetone-water (75/25; v/v). An appropriate amount of the internal standard solution was added, and each solvent extraction was performed by stirring for 3 h under a nitrogen atmosphere at room temperature. The combined supernatants will be evaporated at < 30 °C to remove organic solvents. Then the extracts were fractionated by chromatography on Waters C18 Sep-Pak cartridge according to the method described by Sun et al. [24] with a few modifications. Briefly, each extract (about 50 ml) was passed through the preconditioned Sep-Pak cartridge. Sugar and some phenolic acids were eliminated by elution with 200 ml water. After the cartridge was dried with N2, elutions were carried out first with 150 ml ethyl acetate to elute monomers and oligomers, and then with 100 ml methanol to wash the cartridge. The ethyl acetate elution was evaporated to dryness and redissolved with 2 ml methanol-water (50:50, v/v). 2.4. UHPLC-FT-ICR/MS instrumentation and conditions The UHPLC-FT-ICR/MS system consists of an Agilent 1260 UHPLC system equipped with quadruple pump, online degasser, auto-sampler, thermostatically controlled column compartment, diode-array detector (Agilent, USA) and a Bruker Solarix 7.0 T FT-ICR-MS system (Bruker, Germany). Chromatographic separation was achieved on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) protected by a high-pressure column prefilter with gradient elution using a mobile phase composed of 0.1% formic acid in water (solvent A) and methanol (solvent B) at 35 °C. The gradient eluent at a flow rate of 0.3 ml/min was programmed as follows: t [min]/B [%] 0/2, 6/2, 8/4, 14/8, 18/10, 20/10, 22/12, 26/12, 28/14, 34/20, 38/26, 42/32. After each analysis, a wash step of 5 min with 100% B was executed. The detection wavelength was set at 280 nm and the injection volume was 1 μl, 0.5 μl and 0.2 μl for grape skin, stem and seed, respectively. The FT-ICR/MS conditions were as follows: a nebulizer gas pressure of 4.0 bar, a dry gas flow rate of 10.0 L/min, a capillary voltage of −3.5 kV, an ion flight time of 0.8 s, an ion accumulation time of 0.2 s

2. Experimental 2.1. Samples, chemicals and reagents Grape pomace (about 1 kg) of the variety Vidal (Vitis vinifera cv.) from the 2015 harvest was collected immediately after ice-grape 202

Journal of Chromatography B 1068–1069 (2017) 201–209

L. Luo et al.

and a transfer capillary temperature of 250 °C. Full-scan MS data was acquired over an m/z range of 100–1500, and the collision energy was set at 6 V.

The RSDs were used to evaluate the sample stability.

2.5. UPLC-QqQ/MS instrumentation and conditions

All statistical analyses with qualitative and quantitative analysis of phenolic compounds in Vidal grape skin, seed and stem were performed using SPSS (Version 22.0, Chicago, IL, USA). The comparison of means was determined by one-way analysis of variance (ANOVA) with Duncan’s multiple range test. The results were reported as means ± standard deviation (SD).

2.7. Statistical analysis

UPLC-QqQ/MS analysis was performed on a triple-quadrupole tandem mass spectrometer (Waters Xevo TQ-S mass spectrometer, Waters, Micromass MS Technologies, Manchester, UK) coupled to a Waters Acquity UPLC system (Waters, Milford, MA, USA). Chromatographic separations were carried out on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) protected by a highpressure column prefilter with gradient elution using a mobile phase composed of 0.1% formic acid in water (solvent A) and methanol (solvent B) at 35 °C. The gradient eluent at a flow rate of 0.3 ml/min was programmed as follows: t [min]/B [%] 0/2, 6/2, 8/4, 14/8, 18/10, 20/10, 22/12, 26/12, 28/14, 34/20, 38/26, 42/32. After each analysis, a wash step of 5 min with 100% B was executed. The injection volume was 1 μl, 0.5 μl and 0.2 μl for grape skin, stem and seed, respectively. The detection of phenolic composition was operated in the multiple reaction monitoring (MRM) mode using electrospray negative ionization (ESI−). The collision energies (CE) and source cone voltages (CV) were optimized for eight standards. The quantitative parameters are shown in Table 1.

3. Results and discussion 3.1. Optimization of UPLC conditions The optimization of the new UPLC method of phenolic composition in Vidal grape pomace has been carried out. Different mobile phase (including acetonitrile:water, methanol:water, methanol-formic acid:water, and acetonitrile-formic acid:water), flow rate (0.1, 0.3 and 0.5 ml/min) and column temperature (25, 35, and 40 °C) were determined and compared. The results showed that the shortest analysis time and the best resolution were achieved when the column temperature was 35 °C, the flow rate of mobile phase was 0.3 ml/min and a gradient elution mode composed of 0.1% formic acid in water (solvent A) and methanol (solvent B) was programmed as follows: t [min]/B [%] 0/2, 6/2, 8/4, 14/8, 18/10, 20/10, 22/12, 26/12, 28/14, 34/20, 38/ 26, 42/32.

2.6. Method validation 2.6.1. Linearity, limit of detection and limit of quantification The calibration curves were established by injecting each working solution thrice. All calibration curves were constructed from the peak area ratio (standards versus I.S.) versus their concentration. The limit of detection (LOD) and the limit of quantification (LOQ) were evaluated at the concentrations that generated peaks with signal-to-noise values (S/ N) of 3 and 10, respectively.

3.2. Optimization of MS conditions In order to develop an accurate and sensitive qualitative and quantitative method, the MS (FT-ICR/MS) and the MS/MS (QqQ/MS) fragmentation patterns were investigated. First, The FT-ICR/MS spectra in positive and negative ion modes were determined and compared. The results showed that the negative ion mode was more selective and sensitive for phenolic compounds in Vidal grape pomace. So the negative ion mode was used in FT-ICR/MS and QqQ/MS analysis. For the optimization of MRM condition in QqQ/MS, the parameters of cone voltage (CV) and collision energy (CE) were optimized to get the highest relative abundance of precursor and product ions. The quantitative parameters are shown in Table 1.

2.6.2. Precision, repeatability and recovery The precision of the developed method was determined by the intraand inter-day variations. For intra-day test, the samples were analyzed for six times within the same day, while for inter-day test, the samples were examined in triplicate for consecutive three days. The relative standard deviations (RSDs) were calculated as the measure of precision. To determine the repeatability, six replicates of the same samples were prepared and analyzed. Recovery was used to further evaluate the accuracy of the method. A known amount of the 8 standards were added into a 20 g powder of the same samples in sextuplicate, and then extracted and analyzed with the same procedures.

3.3. Identification of individual phenolic compounds in vidal grape pomace Phenolic composition of skin, stem and seed of Vidal grapes was analyzed with two complementary FT-ICR and QqQ instruments to determine structures based on fragmentation patterns using FT-ICR in EIC mode and QqQ in PIS mode. In total, 35 phenolic compounds were identified, which include 3 hydroxybenzoic acid, 3 hydroxycinnamic acid, 2 hydroxycinnamic tartaric esters, 17 flavanols and 10 flavonols are depicted in Fig. 1 and listed in Table 2 along with their retention time, molecular formula, calculated and observed masses and mass errors of each phenolic compound investigated. Thus, in order to obtain the detailed phenolic composition in grape pomace in Vidal grape, a precise characterization of Vidal grape identified by FT-ICR and QqQ MS was performed.

2.6.3. Stability To confirm the stability, the same sample was stored at room temperature and analyzed by replicate injection at 0, 2, 4, 8, 16 and 24 h. Table 1 Retention time (tR), MRM parameters, cone voltage (CV) and collision energy (CE) for each standard measured. Standards

RT(min)

Precursor ion (m/z)

Product ion (m/z)

CV (V)

CE (V)

p-Coumaric acid Gallic acid Ferulic acid Catechin Epicatechin Epicatechin gallate Astragalin Isoquercitrin I.S.

15.66 1.22 13.79 10.35 18.39 28.29

163.07 169.05 193.02 289.06 289.07 441.08

119.21 125.17 134.18 245.11 244.92 289.28

15 24 15 57 47 37

12 14 14 14 12 30

40.05 36.44 3.41

447.15 463.12 296.01

284.07 300.03 268.77

5 26 18

26 26 20

3.3.1. Hydroxybenzoic acid and its derivatives Gallic acid (m/z 169.01436, peak 1) was the first compound to elute and was identified in all of the skin, seed and stem extracts of Vidal grape pomace, the product ion scan (PIS) of the deprotonated molecule 169.01436 [M-H]− showed the typical loss of CO2 (44 amu), giving an ion at m/z 125 [M-H-44]− as the characteristic fragment. This compound was confirmed by comparison with the calculated mass error (0.52 mDa) and standard compound. Peak 2 with [M-H]− ions at m/z 493.12048 was identified as gallic 203

Journal of Chromatography B 1068–1069 (2017) 201–209

L. Luo et al.

Fig. 1. UHPLC-FT-ICR/MS total ion chromatograms of phenolic compounds identified in seed (a) and skin (b) of Vidal grape. Peak identification is shown in Table 2. (1. Gallic acid, 2. Gallic acid dihexose, 3. Gallic acid hexose, 4. Procyanidin B3, 5. Catechin, 6. Procyanidin B4, 7. trans-fertaric acid, 8. cis-fertaric acid, 9. Procyanidin B1, 10. Procyanidin trimer Ⅰ, 11. Procyanidin trimer Ⅱ, 12. Coumaric acid-O-hexoside, 13. Procyanidin trimer Ⅲ, 14. p-Coumaric acid, 15. Procyanidin B2, 16. Procyanidin B-gallate Ⅰ, 17. Epicatechin, 18. Procyanidin B-gallateⅡ, 19. Procyanidin trimer Ⅳ, 20. Procyanidin B-gallate Ⅲ, 21. Ferulic acid, 22. Procyanidin B7, 23. Procyanidin trimer Ⅴ, 24. Epicatechin gallate, 25. Procyanidin Bgallate Ⅳ, 26. Quercetin-3-O-galactoside, 27. Quercetin-3-O-glucuronide, 28. Quercetin-3-O-glucoside, 29. Quercetin-3-O-rutinoside, 30. Kaempferol-3-Ogalactoside, 31. Dihydrokaempferol-3-O-glucoside, 32. Kaempferol-3-O-glucuronide, 33. Dihydroquercetin-3-O-rhamnoside, 34. Kaempferol-3-Oglucoside, 35. Kaempferol-3-O-rutinoside). < span class="xps_Image" > gr1

Table 2 List of compounds identified in three fractions (skin, stem and seed) of Vidal grape. Compounds Hydroxybenzoic acid and its derivatives 1. Gallic acida 2. Gallic acid dihexose 3. Gallic acid hexose Hydroxycinnamic acid and its derivatives 14. p-Coumaric acida 12. Coumaric acid-O-hexoside 21. Ferulic acida Hydroxycinnamic tartaric esters 7. trans-fertaric acid 8. cis-fertaric acid Flavanols 5. Catechina 17. Epicatechina 24. Epicatechin gallatea 9. Procyanidin B1 15. Procyanidin B2 4. Procyanidin B3 6. Procyanidin B4 22. Procyanidin B7 10. Procyanidin trimer Ⅰ 11. Procyanidin trimer Ⅱ 13. Procyanidin trimer Ⅲ 19. Procyanidin trimer Ⅳ 23. Procyanidin trimer Ⅴ 16. Procyanidin B-gallate Ⅰ 18. Procyanidin B-gallateⅡ 20. Procyanidin B-gallate Ⅲ 25. Procyanidin B-gallate Ⅳ Flavonols 30. Kaempferol-3-O-galactoside 34. Kaempferol-3-O-glucoside* 31. Dihydrokaempferol-3-O-glucoside 33. Dihydroquercetin-3-O-rhamnoside 32. Kaempferol-3-O-glucuronide 26. Quercetin-3-O-galactoside 28. Quercetin-3-O-glucoside* 27. Quercetin-3-O-glucuronide 35. Kaempferol-3-O-rutinoside 29. Quercetin-3-O-rutinoside a

tR (min)

Fractions

[M-H]− (FT-ICR)

MS/MS (QqQ)

Theoretical mass

Mass error (ppm)

Formula

1.20 1.91 3.44

sk, st, se sk, st, se sk, st, se

169.01436 493.12043 331.06757

125 331, 169 169, 125

169.01425 493.11989 331.06707

−0.52 −1.09 −1.52

C7H6O5 C19H26O15 C13H15O10

15.54 14.15 21.67

sk, st, se sk, st, se sk, st, se

163.04012 325.09349 193.05074

119 163, 119 149

163.04007 325.09289 193.05063

−0.73 −1.86 −0.85

C9H8O3 C15H18O8 C10H10O4

11.65 12.18

sk sk

325.05689 325.05692

193, 149 193, 149

325.05651 325.05651

−1.19 −1.26

C14H14O9 C14H14O9

10.25 18.29 28.15 12.23 15.81 8.30 10.29 22.36 12.94 13.41 14.45 20.13 22.76 17.59 18.42 20.97 31.24

sk, st, sk, st, sk, st, sk, st, sk, st, sk, st, sk, st, sk, st, sk, st, sk, st, sk, st, sk, st, sk, st, st, se st, se st, se st, se

se se se se se se se se se se se se se

289.07220 289.07216 441.08325 577.13574 577.13564 577.13590 577.13576 577.13542 865.19870 865.19941 865.19924 865.19922 865.19896 729.14685 729.14688 729.14746 729.14723

245 245 289, 425, 425, 425, 425, 425, 577, 577, 577, 577, 577, 577, 577, 577, 577,

289.07176 289.07176 441.08272 577.13515 577.13515 577.13515 577.13515 577.13515 865.19854 865.19854 865.19854 865.19854 865.19854 729.14611 729.14611 729.14611 729.14611

−1.51 −1.38 −1.20 −1.03 −0.85 −1.30 −1.07 −1.02 −0.19 −1.01 −0.81 −0.78 −0.49 −1.02 −1.06 −1.85 −1.54

C15H14O6 C15H14O6 C22H18O10 C30H26O12 C30H26O12 C30H26O12 C30H26O12 C30H26O12 C45H38O18 C45H38O18 C45H38O18 C45H38O18 C45H38O18 C37H30O16 C37H30O16 C37H30O16 C37H30O16

38.45 40.06 38.61 39.76 39.70 35.09 36.41 35.60 40.64 37.14

sk, sk, st st sk, sk, sk, sk, sk, sk,

st, se st, se

447.09380 447.09378 449.10939 449.10929 461.07290 463.08901 463.08878 477.06778 593.15199 609.14704

285 285 287 303 285 301 301 301 285 301

447.09329 447.09329 449.10894 449.10894 461.07255 463.08820 463.08820 477.06746 593.15119 609.14611

−1.10 −1.15 −0.56 −0.79 −0.77 −1.75 −1.25 −0.67 −1.34 −1.52

C21H20O11 C21H20O11 C21H22O11 C21H22O11 C21H17O12 C21H20O12 C21H20O12 C21H18O12 C27H30O13 C27H30O16

st, st, st, st, st, st,

se se se se se se

Comparison with standard. sk, skin; st, stem; se, seed.

204

169 289 289 289 289 289 289 289 289 289 289 289 289 289 289

Journal of Chromatography B 1068–1069 (2017) 201–209

L. Luo et al.

22) were identified in skin, seed and stem in Vidal grape. The product ion scan of deprotonated ion at 577 showed a Retro-Diels-Alder (RDA) product with a neutral loss of 152 u [M-H-152]− at m/z 425. Fragment at m/z 289 derived from the interflavanic bond cleavage, was also observed (Table 2), and the fragment at m/z 245 was derived from fragment 289 that loss of a CO2. Those five procyanidins were identified by the reference compounds. Procyanidin B1, B2, B3, B4 and B7 were obtained by the previous work [22]. Five procyanidin trimers (m/z 865.19, peak 10, 11, 13, 19 and 23) were also found in all skin, seed and stem in Vidal. The product ion scan at m/z 865 showed interflavanic bond cleavage at m/z 577 and 289. The number of procyanidin trimers was more than other grape [26]. Galloyled procyanidins in Vidal grape pomace were identified as (−)-epicatechin-3-O-gallate (m/ z 441.08325, peak 24) in skin, seed and stem extract and four galloyled procyanidin dimers (m/z 729.14, peak 16, 18, 20 and 25) in seed and stem extract only. The PIS at m/z 729 generated an ion at m/z 577 corresponding to the loss of gallic acid, while the more intense fragment at m/z 289 was due to the loss of a (+)-catechin-gallate or a (−)-epicatechin-gallate unit. And the PIS at m/z 441 showed two fragment ions resulting 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.

acid dihexose and it was the second compound to elute as well as peak 3 with [M-H]− ions at m/z 331.06757 was identified as gallic acid hexose and it was the third compound to elute in skin, seed and stem of Vidal grape. The product ion scan (PIS) of the deprotonated molecule of these two compounds showed a typical MS2 fragmentation pattern [MH–2 × glucose]− and [M-H-glucose]−, which yield product ions at m/z 169, explained by the elimination of two or one glucose unit (162 amu), and further eliminate CO2 (44 amu) giving an ion at m/z 125 [M-Hn × glucose-44]− which is the same as gallic acid. Because of the high saccharinity, gallic acid hexose and dihexose were the most common phenolic acid derivatives in the pamace of Vidal grape. 3.3.2. Hydroxycinnamic acid and its derivatives The skin, seed and stem of Vidal grape extract ion chromatograms of FT-ICR/MS showed two ions at m/z 163.04012 and 325.09349. These deprotonated molecules [M-H]− were tentatively identified as p-coumaric acid (peak 14) and coumaric acid hexoside (peak 12), which were corroborated by product ion scan (PIS) experiment in QqQ/MS showing the predominant ion at m/z 119 (loss of CO2) for p-coumaric acid and m/z 163 (loss of hexose) and 119 (then further loss of CO2) for coumaric acid hexoside. Peak identification was accomplished by comparing MS/ MS fragmentation with reported data obtained by LC-ESI–MS in negative mode [5,13], reference compounds and the calculated mass error (0.73 mDa and 1.86 mDa). Ferulic acid (peak 21) was also identified in grape skin, seed and stem of Vidal: the PIS of the deprotonated molecule (m/z 193.05074) showed an ion at m/z 149 derived from the loss of CO2 (44 amu). Furthermore, the identification was confirmed by the very low mass error (0.85mDa) and standard compound. Garrido & Fernanda’s [25] study suggested that this type of derivatives (hydroxycinnamic acid and its derivatives) was not present in grapes and their derived products (except in Riesling wine) but being tartaric esters or diesters preferentially formed instead (see section 3.3.3). However, in this study, both hydroxycinnamic acid and its derivatives have been found in Vidal grape pomace. Lecce et al. [13] also found p-coumaric acid and coumaric acid hexose in Albariño (Vitis vinifera L.) white grape pomace. Therefore, it is possible that hydroxycinnamic acid and its derivatives only exist in white grape instead of red grape variety.

3.3.5. Flavonols Ten flavonols were identified in Vidal grape pomace including three flavonol-O-glucoside (peaks 28, 31 and 34), two −O-galactoside (peak 26 and 30), two −O-glucuronides (peaks 27 and 32), two −O-rutinoside (peak 29 and 35) and one −O-rhamnoside (33). All the flavonols are −O-glycoside derivatives that arising from cleavage of the glycosidic bond and loss of the sugar moieties [27]. The number of flavonols in Vidal grape pomace was more than Fernão Pires white variety [22] in our previous study. UHPLC-FT-ICR/MS analysis of skin, seed and stem of Vidal variety showed ion at m/z 447.093 (peak 30 and 34) and the retention time at 38.45 and 40.06 min, which were tentatively identified as the deprotonated molecules of kaempferol-3-O-galactoside and kaempferol-3-Oglucoside. The PIS mode showed an intense fragment at m/z 285 typical for kaempferol. Compared with reference standards, peak 30 and 34 were identified as kaempferol-3-O-galactoside and kaempferol-3-Oglucoside, respectively. Similar MS2 experiments in QqQ/MS have been done for kaempferol-3-O-glucuronide (m/z 461.07290, peak 32) and kaempferol-3-O-rutinoside (m/z 593.15199, peak 35), presenting the same fragment at m/z 285 for the loss of glucuronide and rutinoside, respectively. UHPLC-FT-ICR/MS analysis of the stem phenolic extract revealed mass signals at m/z 449.109, corresponding to deprotonated ion for dihydrokaempferol-3-O-glucoside (peak 31) and dihydroquercetin-3-O-rhamnoside (peak 33). The PIS mode for dihydrokaempferol-3-O-glucoside showed the fragment at m/z 287 for dihydrokaempferol losing of glucose. The PIS mode for dihydroquercetin3-O-rhamnoside showed the fragment at m/z 303 for dihydroquercetin losing of rhamnose. The chromatograms of the skin, seed and stem extract of Vidal grape in FT-ICR/MS also showed a deprotonated molecule at m/z 463.08 (peak 26 and 28) in 35.09 and 36.41 min. The PIS of deprotonated ion m/z 463.08 gave a product ion at m/z 301, which suggested the presence of quercetin-3-O-galactoside and quercetin-3-Oglucoside. Compared with reference standards, peak 26 was identified as quercetin-3-O-galactoside and peak 28 was identified as quercetin-3O-glucoside. Similar MS2 experiments in QqQ/MS have been done for quercetin-3-O-glucuronide (m/z 477.06778, peak 27) and quercetin-3O-rutinoside (m/z 609.14704, peak 29), showing the same fragment at m/z 301 (quercetin) for the loss of glucuronide and ruyinoside. The phenolic compounds found in Vidal variety in this study were similar to those reported in previous works with other varieties grown in various parts of the world in general, except only little difference, that is, grape pomace like seeds from other white grape variety (Carménère, Cabernet Sauvignon, Merlot and Cabernet Franc etc.) were

3.3.3. Hydroxycinnamic tartaric esters UHPLC-FT-ICR/MS analysis of skin, seed and stem of Vidal grape showed ion at m/z 325.056 (peak 7 and 8) and the retention time at 11.65 and 12.18 min, which were tentatively identified as deprotonated molecules of isomers of fertaric acid (trans-fertaric acid and cis-fertaric acid). Product ion scan (PIS) of deprotonated ion showed a fragment at m/z 193 attributed to ferulic acid, and an ion at m/z 149 that indicated a loss of CO2 from the free ferulic acid. It was not possible to distinguish the isomers only on the basis of fragments and relative intensities in MS spectra in EIC mode and MS/MS spectra in PIS mode (Table 2) except through comparing with the reference compounds. 3.3.4. Flavanols Reverse phase LC column provided a good baseline resolution for the flavan-3-ols, which consisted of (+)-catechin, (−)-epicatechin, epicatechin-gallate, their condensed product and corresponding galloylated derivatives that exhibited monomeric, dimeric and trimeric degree of polymerization [13]. Except for polymeric procyanidins in Vidal grape pomace, oligomeric procyanidins present in Vidal grape are mainly monomers, dimers and trimers in which the elemental units are linked by C4-C8 interflavanols bonds (B-type). Flavonals were identified in skin, seed and stem of Vdial grape, with mass error below 2 ppm. Compared with reference standards, peak 5 and 17 (m/z 289.072) were identified as (+)-catechin and (−)-epicatechin, respectively, with the loss of CO2 (44 amu) showed MS2 at m/z 245. Up to five procyanidin dimers (m/z 577.135, peak 4, 6, 9, 15 and 205

Journal of Chromatography B 1068–1069 (2017) 201–209

not reported to have the glucosidic compounds, such as gallic acid hexose and dihexose, which may be due to the ice variety of Vidal [28]. However, resveratrol, often considered as an important phenolic compound in grape skin, was not found in Vidal.

1.95 3.66 3.05 3.64 2.45 3.74 2.66 2.47

Stability RSD (%)

L. Luo et al.

2.48 4.12 3.36 3.13 2.69 3.79 2.57 2.46 0.64–82 0.66–85 0.76–97 1.13–145 1.13–145 1.72–221 1.74–224 1.81–232

0.064 0.033 0.076 0.057 0.038 0.086 0.006 0.006

0.021 0.007 0.017 0.011 0.011 0.025 0.002 0.002

1.32 2.46 1.72 1.63 0.95 1.62 1.41 0.75

The validated methods were applied to quantify phenolic compounds in 3 Vidal grape pomace samples, including seeds, skins and stems. A total of 35 phenolic compounds were quantified with the internal standard method based on calibration curves. Procyanidin dimers and trimers were quantified with the calibration curve of catechin. Procyanidin B-gallates were quantified with the calibration curve of epicatechin gallate. Gallic acid hexose and dihexose were quantified with the calibration curve of gallic acid. Coumaric acid-O-hexoside was quantified with the calibration curve of p-Coumaric acid. Fertaric acids were quantified with the calibration curve of ferulic acid. Quercetin derivatives were quantified with the calibration curve of quercetin 3-Oglucoside (astragalin) and kaempferol derivates with kaempferol-3-Oglucoside (isoquercitrin). The typical MRM chromatograms are shown in Fig. 2 and the quantitative results are presented in Table 5. Different quantitative phenolic profile (Table 5) were found in Vidal grape pomace (skin, seed and stem), which showed significant differences (p < 0.05) in their contents of phenolic acid, flavanols, flavonols and thier derivatives. As we can seen from Table 5, the total phenolic content including phenolic acid, flavanols, flavonols and thier derivatives in Vidal grape seeds (36 mg/100 g) was much higher than that in stems (18 mg/100 g) and skins (9 mg/100 g). Though the phenolic content in skins was the lowest (9 mg/100 g), the content of skins in Vidal grape pomace was the highest. As for different kinds of phenolics, in Vidal grape pomace, flavanols were the most abundant phenolics with concentrations ranging from 4 to 36 mg/100 g dry matter, followed by flavonols (0.1–4 mg/100 g), hydroxybenzoic acid (0.03–0.5 mg/100 g) and hydroxycinnamic acid (0.1-0.5 mg/100 g). As for each solid part of Vidal grape pomace, the highest amount of flavanols were found in seeds (36 mg/100 g), followed by stems (15 mg/ 100 g), while skins presented the lowest concentration (4 mg/100 g). On the contrary, flavonols were most abundant in skins (4 mg/100 g), followed by stems (3 mg/100 g), whereas seeds (0.1 mg/100 g) were poor in these compounds. Phenolic acid derivatives (hydroxybenzoic acid and hydroxycinnamic acid) were minority compounds in Vidal grape pomace. Considering individual compounds of flavanols, catechin was the predominant flavanols in all skin, seed and stem of Vidal with no significant differences (P > 0.05) between skins (2.5 mg/100 g) and stems (2.6 mg/100 g), while the content of catechin (11 mg/100 g) in seeds was richer than other phenolics compounds. Epicatechin (9 mg/ 100 g) was the second rich compound in seeds, whereas skins (0.4 mg/

Y = 0.06159X + 0.09182 Y = 0.03773X + 0.03845 Y = 0.02629X + 0.02650 Y = 0.02780X + 0.04028 Y = 0.01736X + 0.02348 Y = 0.00678X + 0.01026 Y = 0.21631X + 0.36317 Y = 0.21006X + 0.33290 p-Coumaric acid Gallic acid Ferulic acid Catechin Epicatechin Epicatechin gallate Astragalin Isoquercitrin

0.9999 0.9998 0.9999 0.9997 0.9999 0.9998 0.9997 0.9998

Inter-day (n = 9) Intra-day (n = 6)

Precision RSD (%) LOD (μg/ml) LOQ (μg/ml) Linear range (μg/ml) Calibration curve

r2

3.5. Quantification of phenolic compounds in vidal grape pomace

Compounds

Table 3 Calibration curve, LOD and LOQ, precision, repeatability and stability of standard compounds.

The calibration curves were constructed with six levels of concentration in triplicate. All the curves exhibited good linearity (r2 > 0.9997) over relatively wide concentration ranges. The lowest LOD and LOQ corresponded to astragalin and isoquercitrin (0.006 and 0.002 μg/ml, respectively) and the highest ones to epicatechin gallate (0.086 and 0.025 μg/ml, respectively). The inter- and intra- variations (RSDs) for 8 standard compounds were less than 4.12 and 2.46%, respectively. The repeatability showed as RSDs was in the varied between 2.50 and 4.32%, and the stability was less than 3.66%. The recoveries range from 93.02 to 106.70% with RSDs less than 3.85%. The above data (Tables 3 and 4) were considered to be satisfactory for the quantification of all the compounds of Vidal samples. The method of UHPLC-FT-ICR/MS and UPLC-QqQ/MS was improved in sensitivity, resolution and speed of assay.

2.50 4.01 3.70 3.54 3.67 4.36 3.04 2.55

Repeatability RSD (%) (n = 6)

3.4. Method validation

206

Journal of Chromatography B 1068–1069 (2017) 201–209

L. Luo et al.

Table 4 Recovery of standard compounds. Compounds

Recovery (n = 9) Original (ng)

Spiked (ng)

Detected (ng)

Recovery (%)

RSD (%)

p-Coumaric acid

11.73

Gallic acid

1.55

Ferulic acid

18.01

Catechin

391.78

Epicatechin

55.88

Epicatechin gallate

118.66

Kaempferol-3-O-glucoside

15.29

Quercetin-3-O-glucoside

54.90

5.75 11.50 17.25 0.88 1.75 2.63 8.82 17.63 26.45 194.50 389.00 583.50 26.33 52.67 79.00 59.83 119.67 179.50 7.88 15.77 23.65 27.67 55.33 83.00

17.32 24.00 29.10 2.38 3.25 4.20 26.91 35.54 44.20 584.12 776.02 979.36 84.03 110.28 130.64 181.02 240.56 296.21 23.64 30.52 39.63 80.64 109.05 135.43

97.22 106.70 100.70 94.32 97.14 100.76 100.91 99.43 99.02 98.90 98.78 100.70 106.91 103.28 94.63 104.23 101.86 98.91 105.96 96.58 102.92 93.02 97.87 97.02

1.86 1.84 1.63 3.25 3.09 3.50 3.01 3.85 2.78 2.36 3.26 1.44 2.42 3.60 1.91 0.92 2.56 1.90 1.34 2.10 1.73 2.55 2.86 0.82

Fig. 2. MRM chromatograms of samples obtained in negative mode for the standard compounds. The peak numbers are in accordance with the compound numbers in Table 2.

glucoside, ranging from 0.1 to 1.1 mg/100 g dry matter and 1 mg to 2 mg/100 g dry matter, respectively. However, quercetin 3-O-glucoside was more abundant in skins (1.2 mg/100 g) than in stem (0.4 mg/ 100 g) and seed extracts (0.1 mg/100 g), while quercetin 3-O-glucuronide was less in skins (1.4 mg/100 g) than in stems (1.9 mg/100 g) and seed (1.9 mg/100 g). This result is different from other reports such as Jara-Palacios et al. [5] and Sagdic et al. [30], which showed both quercetin 3-O-glucoside and quercetin 3-O-glucuronide were richer in skins than in stems and seeds. Other flavonol derivatives were also found in the pomace of Vidal but is lower than quercetin glycosides in concentration, being the most abundant among kaempferol-3-O-glucoside (0.5 mg/100 g) in skins. In our study, dihydrokaempferol-3-Oglucoside and dihydroquercetin-3-O-rhamnoside, which were not reported, were also detected in low concentrations in Vidal grape seeds only. Phenolic acid in Vidal grape including hydroxybenzoic acid and hydroxycinnamic acid were minority in the skins, stems and seeds,

100 g) and stems (0.4 mg/100 g) were poor in this compound with no significant differences (P > 0.05). Procyanidin dimers B1-B7 were found high concentration in Vidal grape seeds, ranging from 1 mg to 5 mg/100 g dry matter. Procyanidin B3 (3 mg/100 g), B4 (5 mg/100 g) and B7 (1 mg/100 g) were also found with high concentration, being B4 and B7 the predominant dimers in stems. The content of procyanidin dimers in Vidal grape skins were low. Furthermore, the unidentified procyanidin trimers and galloyled dimers presented low concentration (< 0.7 mg/100 g) in all pomace of Vidal grape. Zhang et al. [28] pointed out clearly that galloylated procyanidins were confirmed to have much higher antioxidant activities than the non-galloylated ones. Though the amount of galloyled dimers are low in Vidal grape pomace, the number of which showed higher than Zalema [5], Albariño [13]. The flavanols except catechin in Vidal grape skins all showed lower levels than seeds and stems. The main flavonols in the Vidal grape pomace were quercetin derivatives, including quercetin 3-O-glucuronide and quercetin 3-O207

Journal of Chromatography B 1068–1069 (2017) 201–209

L. Luo et al.

flavonols were not detected and in Carménère, Cabernet Sauvignon, Merlot and Cabernet Franc grape, only flavanols was detected [11,29]. For grape skins, the content of some individual flavonols (kaempferol-3O-galactoside, kaempferol-3-O-glucoside, quercetin-3-O-galactoside and quercetin-3-O-glucuronide) in Vidal grape, as shown in Table 5, was higher than Albariño grape (produced in Chile) and BRS Lorena grape (developed in Brazil) [36]. However, Vidal grape presented low concentration of kaempferol and quercetin [11,35]. Through combination of two spectrometric techniques, we were able to identify up to 35 phenolic compounds in Vidal grape pomace, which to our knowledge is the first report for this grapevine variety. We demonstrated FT-ICR/MS is a very useful tool to identify grape phenolic composition because it shows high sensitivity, high resolution and high mass accuracy. Good fits were obtained for all investigated ions, with all mass errors below 2 ppm. The QqQ system was effective for obtaining further identified information about the phenolic composition through PIS experiment. Moreover, QqQ/MS was a powerful system for the quantification of phenolic compounds, whose applicability is demonstrated by validation experiments such as the linearity, repeatability, precision and stability. Therefore, the combination of FT-ICR/ MS and QqQ/MS was quite effective and sensitive to identify and quantify phenolic compounds in grape pomace.

Table 5 Content of phenolic compounds in the different anatomical parts of Vidal grape (mg/ 100 g dry matter). Compounds

Skin

Stem

Seed

Gallic acid Gallic acid hexose Gallic acid dihexose Hydroxybenzoic acid p-Coumaric acid Coumaric acid-Ohexoside Ferulic acid Trans-fertaric acid Cis-fertaric acid Hydroxycinnamic acid Catechin Epicatechin Epicatechin gallate Procyanidin B1 Procyanidin B2 Procyanidin B3 Procyanidin B4 Procyanidin B7 Procyanidin trimer Ⅰ Procyanidin trimer Ⅱ Procyanidin trimer Ⅲ Procyanidin trimer Ⅳ Procyanidin trimer Ⅴ Procyanidin B-gallate Ⅰ Procyanidin B-gallate Ⅱ Procyanidin B-gallate Ⅲ Procyanidin B-gallate Ⅳ Flavanols Kaempferol-3-Ogalactoside Kaempferol-3-Oglucoside Dihydrokaempferol3-O-glucoside Dihydroquercetin-3O-rhamnoside Kaempferol-3-Oglucuronide Quercetin-3-Ogalactoside Quercetin-3-Oglucoside Quercetin-3-Oglucuronide Kaempferol-3-Orutinoside Quercetin-3-Orutinoside Flavonols Total

0.016 0.409 0.089 0.514 0.077 0.216

± ± ± ± ± ±

0.002a 0.002a 0.002a 0.002a 0.002b 0.013a

0.011 0.015 0.004 0.031 0.080 0.119

± ± ± ± ± ±

0.126 0.044 0.036 0.500 2.546 0.383 0.022 0.087 0.146 0.368 0.356 0.087 0.027 0.003 0.011 0.008 0.005 n.d.c

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.002a 0.009 0.009 0.032a 0.087b 0.020b 0.005b 0.007b 0.009b 0.023c 0.011c 0.008c 0.004c 0.001c 0.002b 0.001c 0.001b

0.127 n.d. n.d. 0.326 2.583 0.385 0.845 0.143 0.706 2.573 4.756 1.485 0.426 0.139 0.004 0.092 0.023 0.016

± 0.002a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.002b 0.003c 0.000b 0.002c 0.002b 0.009b

0.006 0.035 0.005 0.046 0.110 0.001

± ± ± ± ± ±

0.001c 0.003b 0.001b 0.004b 0.002a 0.000c

0.015 ± 0.006b n.d. n.d. 0.127 ± 0.007c 11.051 ± 0.608a 8.507 ± 0.066a 0.408 ± 0.048b 3.064 ± 0.082a 5.117 ± 0.091a 2.864 ± 0.051a 2.382 ± 0.045b 1.121 ± 0.099b 0.241 ± 0.019b 0.027 ± 0.003b 0.030 ± 0.006a 0.047 ± 0.008b 0.369 ± 0.056a 0.095 ± 0.009a

0.010b 0.182b 0.025b 0.011a 0.016b 0.018b 0.075b 0.232a 0.042a 0.012a 0.009a 0.001c 0.006a 0.003b 0.008b

n.d.c

0.676 ± 0.022a

0.267 ± 0.022b

n.d.c

0.119 ± 0.002a

0.016 ± 0.001b

n.d.

n.d.

4. Conclusions Our reported a complete, detailed characterization of the phenolic composition of the different solid parts of the Vidal grape. It is the first time that ultrahigh-performance liquid chromatography tandem two complementary techniques, fourier transform ion cyclotron resonance mass spectrometry and triple-quadrupole mass spectrometry, have been applied to allow the phenolic composition to be identified and quantified. Furthermore, an exhaustive characterization of the phenolic composition of the different solid parts of Vidal grape has been studied for the first time. Results confirmed that the developed method was effective, sensitive and suitable for simultaneous determination of the major phenolic compounds in Vidal grape pomace. Through this study, environmental and economical problems caused by these residues generated by winemaking industry could be minimized by the exploitation and valorization of those products. In addition, this study provides useful information for future selection of suitable pomace for extracting potential health-promoting compounds.

0.018 ± 0.003

c

b

4.049 ± 0.116 0.165 ± 0.008a

14.970 ± 0.398 0.043 ± 0.007b

35.625 ± 0.706a 0.006 ± 0.001c

0.506 ± 0.013a

0.104 ± 0.008b

0.003 ± 0.000c

n.d.

n.d.

0.003 ± 0.001

n.d.

n.d.

0.006 ± 0.001

0.060 ± 0.003a

0.051 ± 0.004b

0.007 ± 0.001c

0.278 ± 0.010a

0.119 ± 0.007b

0.007 ± 0.001c

1.160 ± 0.095a

0.388 ± 0.004b

0.059 ± 0.004c

1.378 ± 0.029b

1.877 ± 0.053a

1.877 ± 0.053a

0.001 ± 0.000c

0.020 ± 0.003a

0.010 ± 0.001b

0.003 ± 0.000c

0.014 ± 0.002a

0.010 ± 0.001b

3.551 ± 0.096a 8.613 ± 0.045c

2.598 ± 0.075b 17.925 ± 0.329b

0.119 ± 0.005c 35.916 ± 0.708a

Conflict of interest None. References [1] N. Shitan, Secondary metabolites in plants: transport and self-tolerance mechanisms, Biosci. Biotech. Biochem. 80 (2016) 1283–1293. [2] M.M. Hasan, T. Bashir, H. Bae, Use of ultrasonication technology for the increased production of plant secondary metabolites, Molecules 22 (2017) 1–10. [3] M. Fanzone, F. Zamora, V. Jofre, M. Assof, A. Pena-Neira, Phenolic composition of malbec grape skins and seeds from valle de uco (Mendoza: Argentina) during ripening, Eff. Cluster Thinning J. Agric. Food Chem. 59 (2011) 6120–6136. [4] R. Perestrelo, Y. Lu, S.A.O. Santos, A.J.D. Silvestre, C.P. Neto, J.S. Câmara, S.M. Rocha, Phenolic profile of Sercial and Tinta Negra Vitis vinifera L. grape skins by HPLC–DAD–ESI–MSn: Novel phenolic compounds in Vitis vinifera L. grape, Food Chem. 135 (2012) 94–104. [5] M.J. Jara-Palacios, D. Hernanz, M.L. Escudero-Gilete, F.J. Heredia, Antioxidant potential of white grape pomaces: phenolic composition and antioxidant capacity measured by spectrophotometric and cyclic voltammetry methods, Food Res. Int. 66 (2014) 150–157. [6] M.D. Mossalayi, J. Rambert, E. Renouf, M. Micouleau, J.M. Mérillon, Grape polyphenols and propolis mixture inhibits inflammatory mediator release from human leukocytes and reduces clinical scores in experimental arthritis, Phytomedicine 21 (2014) 290–297. [7] T. Sun, Q.Y. Chen, L.J. Wu, X.M. Yao, X.J. Sun, Antitumor and antimetastatic activities of grape skin polyphenols in a murine model of breast cancer, Food Chem. Toxicol. 50 (2012) 3462–3467. [8] B.S. Sun, T.T. Pinto, M.C. Leandro, J.M. Ricardo-Da-Silva, M.I. Spranger, Transfer of

Each value represents mean (n = 3) ± SD. Values in the same row followed by different letters are significantly different by ANOVA test (p < 0.05).

accounting for 12.5% in skins, 1.9% in stems and 0.5% for seeds of total phenolic contents. Gallic acid hexose (0.4 mg/100 g) was the most abundant non-flavonoid phenolic compound in skins. Furyhermore, trans-fertaric acid and cis-fertaric acid were the only hydroxycinnamic tartaric esters found in skins. Based on the published data about the content of the pehnolic compounds in other grape varieties and other areas, Albariño grape variety (produced in Spain) and Carménère, Cabernet Sauvignon, Merlot and Cabernet Franc grape varieties (produced in Chile) and some varieties from French, Germany and Italy vineyard [31–34] presented higher concentrations than the Vidal grape variety studied in this work, but in Albariño grape seed, hydroxycinnamic acids and 208

Journal of Chromatography B 1068–1069 (2017) 201–209

L. Luo et al.

[9]

[10]

[11]

[12] [13]

[14] [15]

[16]

[17]

[18]

[19]

[20]

[21]

catechins and proanthocyanidins from solid parts of the grape cluster into wine, Am. J. Enol. Vitic. 50 (1999) 179–184. R.R. Tian, G. Li, S.B. Wan, Q.H. Pan, J.C. Zhan, J.M. Li, Q.H. Zhang, W.D. Huang, Comparative study of 11 phenolic acids and five flavan-3-ols in cv. Vidal: impact of natural icewine making versus concentration technology, Aust. J. Grape Wine R. 15 (2009) 216–222. G.J. Soleas, G.J. Pickering, Influence of variety wine style, vintage and viticultural area on selected chemical parameters of Canadian Icewine, J. Food Agric. Envirron. 5 (2007) 97–101. I. Dami, S. Ennahli, D. Scurlock, A five-year study on the effect of cluster thinning and harvest date on yield, fruit composition, and cold-hardiness of ‘Vidal blanc’ (Vitis spp.) for ice wine production, Hortscience 48 (2013) 1358–1362. A.J. Bowen, A.J. Reynolds, Aroma compounds in Ontario Vidal and Riesling icewines. I. effects of arvest date, Food Res. Int. 76 (2015) 540–549. G.D. Lecce, S. Arranz, O. Jáuregui, A. Tresserra-Rimbau, P. Quifer-Rada, R.M. Lamuela-Raventós, Phenolic profiling of the skin: pulp and seeds of Albariño grapes using hybrid quadrupole time-of-flight and triple-quadrupole mass spectrometry, Food Chem. 145 (2014) 874–882. G. Giovinazzo, F. Grieco, Functional properties of grape and wine polyphenols, Plant Foods Hum. Nutr. 70 (2015) 454–462. D. Blanco-Vega, S. Gómez-Alonso, I. Hermosín-Gutiérrez, Identification: content and distribution of anthocyanins and low molecular weight anthocyanin-derived pigments in Spanish commercial red wines, Food Chem. 158 (2014) 449–458. Z. Wei, Y. Pan, L. Li, Y. Huang, X. Qi, M. Luo, Y. Zu, Y. Fu, Simultaneous determination of phenolic compounds in Equisetum palustre L by ultra high performance liquid chromatography with tandem mass spectrometry combined with matrix solid-phase dispersion extraction, J. Sep. Sci. 37 (2014) 3045–3051. L. Li, S. Zhang, Y. Cui, Y. Li, L. Luo, P. Zhou, B. Sun, Preparative separation of cacao bean procyanidins by high-speed counter-current chromatography, J. Chromatogr. B 1036–1037 (2016) 10–19. F. Han, Y. Li, X. Zhang, A. Song, H. Zhu, R. Yin, A pilot study of direct infusion analysis by FT-ICR MS for rapid differentiation and authentication of traditional Chinese herbal medicines, Int. J. Mass spectrom. 403 (2016) 62–67. M. Qian, H. Zhang, L. Wu, N. Jin, J. Wang, K. Jiang, Simultaneous determination of zearalenone and its derivatives in edible vegetable oil by gel permeation chromatography and gas chromatography–triple quadrupole mass spectrometry, Food Chem. 166 (2015) 23–28. F. Sánchez-Rabaneda, O. Jáuregui, R.M. Lamuela-Raventós, F. Viladomat, J. Bastida, C. Codina, Qualitative analysis of phenolic compounds in apple pomace using liquid chromatography coupled to mass spectrometry in tandem mode, J. Mass Spectrom. 18 (2004) 553–563. A. Vallverdu-Queralt, O. Jáuregui, A. Medina-Remón, C. Andres-Lacuéva, M. Lamuela-Raventós, Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap

[22]

[23]

[24]

[25] [26]

[27]

[28]

[29]

[30]

[31] [32]

[34]

[35] [36]

209

mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry, Rapid Commun. Mass Spectrom. 24 (2010) 2986–2992. L. Luo, Y. Cui, S. Zhang, L. Li, Y. Li, P. Zhou, B. Sun, Preparative separation of grape skin polyphenols by high-speed counter-current chromatography, Food Chem. 212 (2016) 712–721. Y. Cui, Q. Li, M. Zhang, Z. Liu, W. Yin, W. Liu, X. Chen, K. Bi, L.C.M.S. Determination, Pharmacokinetics of p-Coumaric acid in rat plasma after oral administration of p-Coumaric acid and freeze-Dried red wine, J. Agric. Food Chem. 58 (2010) 12083–12088. B.S. Sun, M.C. Leandro, J.M. Ricardo-da-Silva, M.I. Spranger, Separation of grape and wine proanthocyanidins according to their degree of polymerization, J. Agric. Food Chem. 46 (1998) 1390–1396. G. Jorge, B. Fernanda, Wine and grape polyphenols — A chemical perspective, Food Res. Int. 54 (2013) 1844–1858. S. Zhang, L. Li, Y. Cui, L. Luo, Y. Li, P. Zhou, B. Sun, Preparative high-speed countercurrent chromatography separation of grape seed proanthocyanidins according to degree of polymerization, Food Chem. 219 (2017) 399–407. N. Castillo-Muñoz, S. Gómez-Alonso, E. García-Romero, I.J. Hermosín-Gutiérrez, Flavonol profiles of Vitis vinifera white grape cultivars, J. Food Compos. Anal. 23 (2010) 699–705. S. Zhang, Y. Cui, L. Li, Y. Li, P. Zhou, L. Luo, B. Sun, Preparative HSCCC isolation of phloroglucinolysis products from grape seed polymeric proanthocyanidins as new powerful antioxidants, Food Chem. 188 (2015) 422–429. E. Obreque-Slier, R. López-Solís, L. Castro-Ulloa, C. Romero-Díaz, A. Peña-Neira, Phenolic composition and physicochemical parameters of Carménère, Cabernet Sauvignon: merlot and Cabernet Franc grape seeds (Vitis vinifera L.) during ripening, LWT-Food Sci. Technol. 48 (2012) 134–141. O. Sagdic, I. Ozturk, G. Ozkan, H. Yetim, L. Ekici, M.T. Yilmaz, RP-HPLC–DAD analysis of phenolic compounds in pomace extracts from five grape cultivars: evaluation of their antioxidant, antiradical and antifungal activities in orange and apple juices, Food Chem. 126 (2011) 1749–1758. P. Rondeau, F. Gambier, F. Jolibert, N. Brosse, Compositions and chemical variability of grape pomaces from French vineyard, Ind. Crop Prod. 43 (2013) 251–254. A. Kammerer, R. Claus, A. Carle, Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS, J. Agric. Food Chem. 52 (2004) 4360–4367. A. Gismondi, G.D. Marco, L. Canuti, A. Canini, Antiradical activity of phenolic metabolites extracted from grapes of white and red Vitis vinifera L cultivars, Vitis 56 (2017) 19–26. S. Impei, A. Gismondi, L. Canuti, A. Canini, Metabolic and biological profile of autochthonous Vitis vinifera L ecotypes, Food Funct. 6 (2015) 1526–1538. M.T. Barcia, P.B. Pertuzatti, S. Gómez-Alonso, H.T. Godoy, I. Hermosín-Gutiérrez, Phenolic composition of grape and winemaking by-products of Brazilian hybrid cultivars BRS Violeta and BRS Lorena, Food Chem. 159 (2014) 95–105.