Food Chemistry 136 (2013) Contents lists available at SciVerse ScienceDirect. Food Chemistry

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1 Food Chemistry 136 (2013) Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: Characterization and identification of the chemical constituents from tartary buckwheat (Fagopyrum tataricum Gaertn) by high performance liquid chromatography/photodiode array detector/linear ion trap FTICR hybrid mass spectrometry Qiang Ren, Caisheng Wu, Yan Ren, Jinlan Zhang Peking Union Medical College & State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing , PR China article info abstract Article history: Received 3 June 2012 Received in revised form 7 September 2012 Accepted 14 September 2012 Available online 23 September 2012 Keywords: Tartary buckwheat HPLC-PDA/LTQ-FTICRMS Phenlypropanoid glycosides Flavonoids In recent years tartary buckwheat has become popular healthful food due to its antioxidant, antidiabetic and antitumor activities. However, its chemical constituents have not yet been fully characterized and identified. In this paper, a novel high performance liquid chromatography coupled with photodiode array detector and linear ion trap FTICR hybrid mass spectrometry (HPLC-PDA/LTQ-FTICRMS) method was established to characterize and identify a total of 36 compounds by a single run. The retention time, maximum UV absorption wavelength, accurate mass weight and characteristic fragment ions were collected on line. To confirm the structures, 11 compounds were isolated and identified by MS and NMR experiments. 1, 3, 6, 6 0 -tetra-feruloyl sucrose named taroside was a new phenlypropanoid glycoside, together with 3, 6-di-p-coumaroyl-1, 6 0 -di-feruloyl sucrose, 1, 6, 6 0 -tri-feruloyl-3-p-coumaroyl sucrose, N-transferuloyltyramine and quercetin-3-o-[b-d-xyloxyl-(1? 2)-a-L-rhamnoside] were isolated for the first time from the Fagopyrum species. The research enriched the chemical information of tartary buckwheat. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Tartary buckwheat belongs to the Polygonaceae family, which has two main species, including common buckwheat (Fagopyrum esculentum Moench) and tartary buckwheat (Fagopyrum tataricum Gaertn). Tartary buckwheat and common buckwheat originated from the southwest China and the Himalayan hills (Ohnishi, 1998). In recent years tartary buckwheat has gained much attention, due to its benefits for human health. In food processing, tartary buckwheat has been used to make various healthful foods such as noodles, herb tea and crackers in Asian countries. Not only tartary buckwheat leaves but also sprouts are consumed as nutritional vegetable. Therefore, tartary buckwheat is recognized as a healthy food. The pharmacological investigations demonstrated that tartary buckwheat had a variety of pharmacological activities such as antioxidant activity, which was determined by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability (Kim, Tsao, Yang, & Cui, 2006; Kim et al., 2008; Liu, Chen, Yang, & Chiang, 2008; Wang, Liu, Gao, Parry, & Wei, 2009), antitumor activity of tartary buckwheat protein against human mammary cancer cell Bcap37 (Guo, Zhu, Corresponding author. Tel.: ; fax: address: zhjl@imm.ac.cn (J. Zhang). Zhang, & Yao, 2007) and antidiabetic activity of tartary buckwheat bran extract being investigated through male KK-Ay mice (type 2 diabetic) and C57BL/6 mice (the control) (Yao et al., 2008). Li et al. reported that three flavonoids such as quercetin, isoquercetin and rutin from tartary buckwheat bran were effective inhibitors against a-glucosidase (Li, Zhou, Gao, Bian, & Shan, 2009). In addition, tartary buckwheat could reduce the level of total cholesterol, lower the concentration of blood glucose and regulate the lipid profile (Wang et al., 2009; Yao et al., 2008). Many phytochemical investigations mainly focused on common buckwheat. However, there were only a few systematic investigations about tartary buckwheat. Previous studies on tartary buckwheat revealed that some types of compounds had been identified such as flavonoids (rutin, quercetin and quercitrin), C-glycosylflavones (orientin, isoorientin, vitexin and isovitexin) (Kim et al., 2007a,b; Kim et al., 2009), flavan-3-ol monomers (catechin and epicatechin) (Kim et al., 2009), organic acids (caffeic, ferulic, gallic, chlorogenic, (+)-osbeckic, 5-hydroxymethyl-2-furoic, protocatechuic and p-hydroxybenzoic acids) (Kim et al., 2007a,b; Kim et al., 2009; Matsui, Kudo, Tokuda, Matsumoto, & Hosoyama, 2010), anthocyanins (cyanidin-3-o-glucoside and cyanidin-3- O-rutinoside) (Kim et al., 2007a,b), D-chiro-inositol and transreveratrol (Němcová, Zima, Barek, & Janovská, 2011; Yang & Ren, 2008) were reported on the occurrence of tartary buckwheat. It /$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.

2 1378 Q. Ren et al. / Food Chemistry 136 (2013) was just recently that seven phenylpropanoid glycosides including tatarisides A-G had been reported in tartary buckwheat root (Zheng et al., 2012). Hence, it is necessary to extensively characterize and identify the chemical constituents of tartary buckwheat. High performance liquid chromatography coupled with mass spectrometry plays an important role in the analysis for the complex and minor constituents of crude herb extract. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides excellent mass accuracy for the determination of elemental composition and high mass resolution for the difficult chromatographic separation. The LTQ MS can provide multi-stage mass analysis (MS n ), which is automatically carried out by means of data dependent scan experiments. The FTICR-MS collects full scan MS data, then chooses the most intense parent ion or the parent ions of interest for multi-stage mass fragmentation. The target ions are fragmented. The cycle is repeated throughout the duration of the acquisition and provides a great deal of data, which is best used for structural elucidation (Vallverdú-Queralt, Jáuregui, Di Lecce, Andrés-Lacueva, & Lamuela-Raventós, 2011). The objective of current study is to develop a comprehensive analysis method for the chemical profile of tartary buckwheat and identify the major and minor constituents by HPLC-PDA/LTQ- FTICRMS. In this paper, accurate mass and multiple-stage mass data were collected on line. Then the structures of major constituents were confirmed by spectroscopic methods and their characteristic fragmentation patterns were summarized. The minor chemical constituents were identified on the basis of their accurate mass and characteristic fragmentation behavior of known compounds. 2. Materials and methods 2.1. Materials Chlorogenic acid, rutin, catechin, and epicatechin were purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Quercetin-3-O-b-D-glucoside (peak 19), kaempferol-3-o-b-d-galactoside (peak 21), kaempferol-3-o-b-d-glucoside (peak 22), quercetin-3-o-b-d-galactoside (peak 17), quercetin-3-o-a-l-rhamnoside (peak 23), N-trans-feruloyltyramine (peak 26), quercetin-3-o-[b-d-xyloxyl-(1? 2)-a-L-rhamnoside] (peak 24), 1, 3, 6-tri-p-coumaroyl-6 0 -feruloyl sucrose (peak 33), 3, 6-di-p-coumaroyl-1, 6 0 -di-feruloyl sucrose (peak 34), 1, 6, 6 0 -tri-feruloyl-3-pcoumaroyl sucrose (peak 35), 1, 3, 6, 6 0 -tetra-feruloyl sucrose (peak 36) were isolated from tartary buckwheat in our laboratory. The purity of reference substances were determined to be higher than 98% by HPLC. These structures were confirmed by their MS, 1D NMR and 2D NMR. The structures are shown in Fig. 1. The total plant of tartary buckwheat (F. tataricum Gaertn) with root, stem, and leaf was collected in the wild from HuiZe, Yunnan province by Professor Lin Ma during in July The collected plant material was dried in the shade. The voucher specimen (No ) was deposited at the Institute of Materia Medica, Chinese Academy of Medical Sciences, China. Acetonitrile of LC/MS reagent grade was supplied by Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). Deionized water was purified by Millipore water purification system (Millipore, MA, USA). Analytical grade dichlormethane, methanol and acetic acid were purchased from Beijing Chemical Corporation (Beijing, China). Sephadex LH-20 was obtained from GE Healthcare (made in Sweden). Silica gel ( mesh) was supplied by Qingdao Marine Chemical Company (Qingdao, People s Republic of China) Sample preparation The dry tartary buckwheat was cut into small pieces. Accurately 1.0 g of tartary buckwheat was weighed and transferred into a glass scintillation vial with 5 ml of 70% aqueous ethanol, which was treated with ultrasonication for 30 min at ambient temperature. The extract was centrifuged at 4500 rpm for 10 min, then filtered through a 0.22 lm filter and analyzed by HPLC-PDA/LTQ-FTICRMS Optimization of extract methods To achieve good extraction efficiency, sample preparation was optimized. Two kinds of routine extract methods such as ultrasonication and reflux were investigated. Ultrasonication (100 W, 40 khz) was in 5 ml different extraction solvents including methanol, 70% aqueous methanol, ethanol and 70% aqueous ethanol with extraction time 30 min at ambient temperature. Reflux extraction in a water bath (45 C) for 30 min with 5 ml four kinds of different solvents. Ultrasonic extraction was selected, because it was more simple and faster than reflux. Then, various extraction solvents including methanol, 70% aqueous methanol, ethanol and 70% aqueous ethanol were tried. 70% aqueous ethanol could extract a wide variety of compounds, which was of great benefit to profile the chemical constituents of tartary buckwheat HPLC-PDA/LTQ-FTICRMS analysis HPLC-PDA analysis was performed on a Finnigan Surveyor LC plus system (Thermo Fisher, Co. Ltd., San Jose, CA USA), equipped with a surveyor MS pump plus and surveyor autosampler. Chromatographic separation was carried out on a Kinetex C 18 reverse phase column ( mm, 2.6 lm, Phenomenex). PDA detector recorded from 200 to 400 nm. The detection monitored simultaneously at 210 nm, 280 nm and 320 nm. The mobile phase consisted of water containing 0.1% acetic acid (v/v) (A) and acetonitrile (B). Gradient elution was applied as follows: 0 20 min for 6 16% B; min for 16 20% B; min for 20 33% B; min for 33 38% B; min for % B; min for 100% B, followed by re-equilibration of the column for 8 min. The flow rate was set as 0.5 ml/min, and the column temperature was maintained at 35 C. The injection volume was 2 ll. The FTICR-MS full scan and multi-stage mass experiments were carried out on Finnigan LTQ FT (Thermo Fisher, Co. Ltd., San Jose, CA USA), which contained two kinds of gas that ultra high purity helium was used as collision gas and high purity nitrogen as nebulizer gas. The optimized electrospray ion source parameters were as follows: ion spray voltage at 3.5 kv, capillary temperature at 250 C, capillary voltage at 30 V, sheath gas flow rate at 45 (arbitrary units), auxiliary gas flow rate at 10 (arbitrary units), sweep gas flow rate at 3 (arbitrary units) and tube lens offset voltage at 90 V. Positive ionization gained a better result than negative ionization. Most of the peaks were detected, which signals corresponded to the protonated ion [M+H] + or the sodium adduct ion [M+Na] +. The effluent from HPLC system was split (1:1) and sprayed into mass spectrometer with an electrospray interface operating in positive ionization for full scan in a m/z range of , with 3 microscans and a maximum ion injection time of 200 ms. The data was acquired and analyzed by means of Xcalibur 2.0 software (Thermo Fisher Scientific, Waltham, MA) Extraction and isolation of tartary buckwheat The dry tartary buckwheat (1600 g) was extracted with 70% aqueous ethanol (3.5 L 3) and then evaporated to dryness under vacuum at 40 C. The crude extract (254 g) was resolved in water (1.5 L), which partitioned respectively with petroleum ether (1.5 L 3), acetoacetate (EtOAc) (1.5 L 3) and n-butanol (n- BuOH) (1.5 L 3), respectively. After analysis of different partitioned extracts by TLC and HPLC-MS, the EtOAc extract (20 g)

3 Q. Ren et al. / Food Chemistry 136 (2013) Fig. 1. Structures of the isolated compounds from tartary buckwheat. (1) quercetin-3-o-b-d-galactoside, (2) quercetin-3-o-b-d-glucoside, (3) kaempferol-3-o-b-dgalactoside, (4) Kaempferol-3-O-b-D-glucoside, (5) quercetin-3-o-a-l-rhamnoside, (6) quercetin-3-o-[b-d-xyloxyl-(1? 2)-a-L-rhamnoside], (7) N-trans-feruloyltyramine, (8) 1, 3, 6 -tri-p-coumaroyl-6 0 -feruloyl sucrose, (9) 3, 6-di-p-coumaroyl-1,6 0 -di-feruloyl sucrose, (10) 1, 6, 6 0 -tri-feruloyl-3-p-coumaroyl sucrose, (11) 1, 3, 6, 6 0 -tetra-feruloyl sucrose. enriched the target components such as flavonoids and phenylpropanoid glycosides and was subjected to mesh silica gel column ( mm i.d.) eluting with CH 2 Cl 2 and MeOH (99:1? 0:100), and then gained eight fractions (A H). The all fractions were monitored by TLC and HPLC-MS. The fraction D, E, F, G and H concentrated the target compounds were pooled for further isolation, and the fraction A C were laid aside for later studies. Fraction D (1.8 g) was further applied to Sephadex LH-20 column ( mm i.d.) and eluted with CH 2 Cl 2 and MeOH (1:1) to yield 60 sub-fractions. The sub-fractions from 21 to 43 were pooled and concentrated, then subjected to preparative HPLC ZOR- BAX Eclipse XDB-C 18 ( mm, 5 lm) column. The column was eluted by CH 3 CN and H 2 O containing 5% MeOH (19:81) with flow rate at 3.0 ml/min, detection at 320 nm. The peak 26 (20.6 mg) was obtained. Fraction E (7.0 g) was subjected to chromatography on silica gel ( mesh) column ( mm i.d.) and eluted with CH 2- Cl 2 and MeOH (9:1) to yield 30 sub-fractions. Sub-fractions from 6 to 21 (2.35 g) were pooled and concentrated, then successively separated on Sephadex LH-20 column for further purification with CH 2 Cl 2 -MeOH (1:1) and gained 55 sub-fractions, and then used ZORBAX Eclipse XDB-C 18 ( mm, 5 lm) column with the flow rate at 4.0 ml/min, detection at 320 nm, eluting with CH 3 CN and H 2 O containing 5% MeOH (32:68), which yielded peak 33 (10.5 mg) from sub-fractions 46 to 50, peak 34 (11.2 mg) from sub-fractions 41 to 43, peak 35 (15.9 mg) from sub-fractions 34 to 38 and peak 36 (14.8 mg) from sub-fractions 22 to 33. Fraction F (2.1 g) was chromatographed on Sephadex LH-20 column ( mm i.d.) with MeOH to afford 66 sub-fractions. Sub-fractions from 19 to 27 were pooled and gained peak 23 (15.1 mg) by preparative HPLC ZORBAX Eclipse XDB-C 18 ( mm, 5 lm) column, a flow rate of 4.0 ml/min, detection at 320 nm, eluting with CH 3 CN and H 2 O containing 5% MeOH (15:85). Fraction G (2.0 g) was separated by Sephadex LH-20 column ( mm i.d.) by elution with MeOH to yield 96 sub-fractions. Sub-fractions from 17 to 24 were pooled and further purified by preparative HPLC Waters Xbridge Prep C 18 ( mm, 10 lm) column with the flow rate at 4.0 ml/min, detection at 320 nm, eluting with CH 3 CN and H 2 O containing 5% MeOH (15:85), then peak 19 (7.1 mg), peak 21 (6.6 mg) and peak 22 (8.3 mg) were obtained. Sub-fractions from 37 to 48 were pooled and gained peak 17 (7.7 mg). Fraction H (1.2 g) was carried out on Sephadex LH-20 column ( mm i.d.) by elution with MeOH to yield 56 sub-fractions. Sub-fractions from 25 to 33 were pooled and further purified by preparative HPLC Waters Xbridge Prep C 18 ( mm, 10 lm) column with flow rate at 3.0 ml/min, detection at 320 nm, eluting with CH 3 CN and H 2 O containing 5% MeOH (19:81), then gained peak 24 (18.6 mg) NMR spectroscopy analysis NMR spectra were obtained on a VNS-600 MHz spectrometer operating at a proton NMR frequency of MHz and carbon NMR frequency of MHz. Samples were added to a 5 mm NMR tube with 450 ll DMSO-d 6. Chemical shifts were given in d (ppm) scale relative to solvent peaks as reference. Acquisition parameters were as follows: spectral width 6000 Hz, relaxation delay 1.0 s, number of scans 3000, pulse width 45, and acquisition time 0.5 s. The experiments were carried out at 25 C. 3. Results and discussion 3.1. General analytical strategy The combination of FTICR mass analyzer, tandem mass spectrometry and PDA detector are capable of providing accurate mass, elemental composition, multiple-stage mass data and maximum UV absorption wavelength for characterization and identification of tartary buckwheat. The isolated reference substances from tartary buckwheat were analyzed by HPLC-PDA/LTQ-FTICRMS. The useful information such

4 1380 Q. Ren et al. / Food Chemistry 136 (2013) as characteristic fragment ions and fragmentation pattern were summarized. Unknown compounds were identified by comparing with the information of reference substances. Moreover, the fragmentation patterns of known compounds were utilized in correlation to propose the structures of unknown compounds Characteristic HPLC-PDA/LTQ-FTICRMS profile of tartary buckwheat Optimization of the chromatographic conditions To achieve good separation and profile comprehensive constituents, the column, mobile phase and elution program were investigated. The different types of columns such as Restek pinnacle II C 18 column ( mm, 5 lm), Shiseido Capcell PAK CR type (1:4) and type (1:50) columns ( mm, 5 lm), ZORBAX series including Extend C 18 column, XDB C 18 column, Eclipse Plus C 18 column, SB-Phenyl column ( mm, 5 lm) and Kinetex C 18 column ( mm, 2.6 lm, Phenomenex) were investigated. Phenlypropanoid glycosides (peak 33, 34, 35 and 36) were difficult to separate. The ZORBAX SB-Phenyl column could provide well separation for them, but poor separation for flavonoids. The Kinetex C 18 reverse phase column exhibited good chromatographic separation for the most constituents and was chosen for analysis. The mobile phase of acetonitrile and water consisting 0.1% acetic acid provided much better resolution for individual peaks. Due to the difference in polarity between phenlypropanoid glycosides and flavonoids, gradient elution was adapted to probe as many constituents as possible in a single run HPLC-PDA/LTQ-FTICRMS profile of tartary buckwheat The extract of tartary buckwheat was analyzed by HPLC-PDA/ LTQ-FTICRMS and the chemical profile was achieved. The representative UV-absorption chromatogram (A), total ion chromatogram (B) and five extract ion chromatograms including quinic acid derivatives (C), flavan-3-ol derivatives (D), flavonol derivatives (E), phenylpropanoid glycosides (F), and nitrogen compounds (G) were obtained in Fig Identification of isolated compounds from tartary buckwheat In order to probe the constituents of tartary buckwheat on the base of their characteristic UV absorbance wavelength and mass data, the major chemical constituents were isolated as reference substances and identified by HRMS and NMR data Structural determination of isolated compounds by NMR Peak 36 was a new compound, which was obtained as a white powder. Its molecular formula was determined to be C 52 H 54 O 23 by ESI-HRMS (m/z [M+Na] +, calculated for (calcd for) C 52 H 54 O 23 Na). The NMR data (Table 1) suggested the existence of four feruloyl groups and a sucrose moiety. Furthermore, the 1 H NMR spectrum of peak 36 showed that eight trans olefinic protons d 7.49 (1H, d, J=15.2 Hz) and d 6.46 (1H, d, J=15.0 Hz); d 7.51 (1H, d, J=15.6 Hz) and d 6.44 (1H, d, J=15.6 Hz); d 7.57 (1H, d, J=15.6 Hz) and d 6.42 (1H, d, J=15.0 Hz); d 7.45 (1H, d, J=15.0 Hz) and d 6.42 (1H, d, J=15.0 Hz) and four feruloyl groups ABX-type signals d 6.69 (1H, d, J=8.4 Hz), 7.01 (1H, dd, J=8.4, 1.2 Hz) and 7.22 (1H, d, J=1.2 Hz); d 6.69 (1H, d, J=7.8 Hz), 6.98 (1H, dd, J=7.8, 1.2 Hz) and 7.22 (1H, d, J=1.2 Hz); d 6.73 (1H, d, J=7.8 Hz), 7.09 (1H, dd, J=7.8, 1.2 Hz) and 7.26 (1H, d, J=1.2 Hz); d 6.71 (1H, d, J=8.4 Hz), 7.04 (1H, dd, J=8.4, 1.2 Hz) and 7.23 (1H, d, J=1.2 Hz). Then the signals for four methoxyls at d 3.76 (3H, s), 3.74 (3H, s), 3.74 (3H, s), 3.78 (3H, s) were exhibited in the 1 H NMR spectrum. The 1 Hand 13 C NMR resonance values were similar to the reported literature (Takasaki, Kuroki, Kozuka, & Konoshima, 2001) for 1, 6, 6 0 -triferuloyl-3-p-coumaroyl sucrose. In the HMBC spectrum, phenyl protons correlated to olefinic carbons, in addition olefinic proton correlated to each ester carbonyl carbon, such as correlations from H-6 0 of glucose to C-9 00 carbonyl, and from H-1, H-3, and H-6 of fructose to C-9 000, C , and C carbonyls, which confirmed the linkage positions. The complete assignment of protons and carbons were established by analyses of 1 H 1 H COSY, HSQC, TOCSY and HMBC spectrum. On the basis of the spectral data, peak 36 was confirmed to be 1, 3, 6, 6 0 -tetraferuloyl sucrose, named taroside. NMR spectroscopic data for peak 36, peak 33, peak 34 and peak 35 are shown in Table 1 and Table 2. By comparing the literature data (Takasaki et al., 2001), peak 33, peak 34 and peak 35 were identified as 1, 3, 6-tri-p-coumaroyl-6 0 -feruloyl sucrose, 3, 6-di-pcoumaroyl-1, 6 0 -di-feruloyl sucrose and 1, 6, 6 0 -tri-feruloyl-3-pcoumaroyl sucrose. Quercetin-3-O-b-D-galactoside (peak 17): yellow powder, ESI- HRMS m/z: [M+H] + (calcd for C 21 H 21 O 12, ). 1 H NMR (DMSO-d 6, 600 MHz): d (1H, br-s, 5-OH), 7.65 (1H, Fig. 2. The representative UV-absorption chromatogram (A), total ion chromatogram (B) and five extract ion chromatograms including quinic acid derivatives (C), flavan-3-ol derivatives (D), flavonol derivatives (E), phenylpropanoid glycosides (F), and nitrogen compounds (G).

5 Q. Ren et al. / Food Chemistry 136 (2013) Table 1 1 H NMR spectral data for peak 33, 34, 35 and 36 in DMSO-d6. Fructose Peak 36 Peak 33 Peak 34 Peak (2H, m) 4.06 (2H, m) 4.09 (2H, m) 4.10 (2H, m) (1H, d, 9.0) 5.43 (1H, d, 8.4) 5.44 (1H, d, 9.0) 5.44 (1H, d, 9.0) (1H, m) 4.38 (1H, m) 4.39 (1H, m) 4.37 (1H, m) (1H, m) 4.08 (1H, m) 4.07 (1H, m) 4.03 (1H, m) (1H, m), 4.50 (1H, m) 4.35 (1H, m), 4.46 (1H, m) 4.35 (1H, m), 4.47 (1H, m) 4.33 (1H, m), 4.49 (1H, m) 4-OH 5.90 (1H, d, 6.0) 5.88 (1H, d, 6.0) 5.88 (1H, d, 6.6) 5.88 (1H, d, 6.0) Glucose (1H, d, 3.6) 5.21 (1H, d, 3.6) 5.23 (1H, d, 3.0) 5.22 (1H, d, 3.6) (1H, m) 3.26 (1H, m) 3.25 (1H, m) 3.27 (1H, m) (1H, m) 3.41 (1H, m) 3.41 (1H, m) 3.41 (1H, m) (1H, m) 3.11 (1H, m) 3.11 (1H, m) 3.12 (1H, m) (1H, m) 4.02 (1H, m) 4.02 (1H, m) 4.05 (1H, m) (1H, m), 4.54 (1H, m) 4.06 (1H, m), 4.51 (1H, m) 4.05 (1H, m), 4.53 (1H, m) 4.07 (1H, m), 4.54 (1H, m) 2 0 -OH 4.90 (1H, d, 6.0) 4.87 (1H, br) 4.88 (1H, br) 4.88 (1H, br) 3 0 -OH 4.88 (1H, d, 4.8) 4.87 (1H, br) 4.88 (1H, br) 4.88 (1H, br) 4 0 -OH 5.33 (1H, d, 5.4) 5.30 (1H, br) 5.33 (1H, d, 4.8) 5.34 (1H, br) Phenylpropanoids glu-6 0 feruloyl feruloyl feruloyl feruloyl (1H, d, 1.2) 7.23 (1H, d, 1.2) 7.22 (1H, d, 1.8) 7.22 (1H, d, 1.2) (1H, d, 8.4) 6.69 (1H, d, 7.8) 6.71 (1H, d, 8.4) 6.71 (1H, d, 7.8) (1H, dd, 8.4, 1.2) 7.02 (1H, dd, 7.8,1.2) 7.20 (1H, dd, 8.4, 1.8) 6.98 (1H, dd, 7.8, 1.2) (1H, d, 15.2) 7.45 (1H, d, 15.0) 7.50 (1H, d, 15.6) 7.45 (1H, d, 15.6) (1H, d, 15.0) 6.30 (1H, d, 16.2) 6.45 (1H, d, 16.2) 6.45 (1H, d, 15.6) 9 00 O Me 3.76 (3H, s) 3.74 (3H, s) 3.79 (3H, s) 3.74 (3H, s) fruc-1 feruloyl p-coumaroyl feruloyl feruloyl (1H, d, 1.2) 7.50 (1H, d, 8.4) 7.22 (1H, d, 1.8) 7.22 (1H, d, 1.2) (1H, d, 7.8) (1H, d, 7.8) 6.73 (1H, d, 7.8) (1H, dd, 7.8, 1.2) 7.50 (1H, d, 8.4) 7.10 (1H, dd, 7.8, 1.8) 7.09 (1H, dd, 7.8, 1.2) (1H, d, 15.6) 7.60 (1H, d, 15.6) 7.58 (1H, d, 15.0) 7.52 (1H, d, 16.2) (1H, d, 15.6) 6.36 (1H. d, 16.2) 6.36 (1H, d, 16.2) 6.42 (1H, d, 15.6) O Me 3.74 (3H, s) 3.74 (3H, s) 3.79 (3H, s) fruc-3 feruloyl p-coumaroyl p-coumaroyl p-coumaroyl (1H, d, 1.2) 7.47 (1H, d, 8.4) 7.40 (1H, d, 8.4) 7.47 (1H, d, 8.4) (1H, d, 8.4) 6.70 (1H, d, 9.0) (1H, d, 7.8) (1H, d, 8.4) 6.70 (1H, d, 9.0) (1H, dd, 7.8, 1.2) 7.47 (1H, d, 8.4) 7.40 (1H, d, 8.4) 7.47 (1H, d, 8.4) (1H, d, 15.6) 7.51 (1H, d, 15.0) 7.51 (1H, d, 15.6) 7.58 (1H, d, 15.6) (1H, d, 15.0) 6.35 (1H, d, 16.2) 6.42 (1H, d, 16.2) 6.36 (1H, d, 16.2) O Me 3.74 (3H, s) fruc-6 feruloyl p-coumaroyl p-coumaroyl feruloyl (1H, d, 1.2) 7.40 (1H, d, 9.0) 7.47 (1H, d, 9.0) 7.24 (1H, d, 1.2) (1H, d, 8.4) 6.69 (1H, d, 8.4) (1H, d, 8.4) 6.68 (1H, d, 8.4) 6.69 (1H, d, 8.4) 6.71 (1H, d, 8.4) (1H, dd, 8.4, 1.2) 7.40 (1H, d, 9.0) 7.47 (1H, d, 9.0) 7.02 (1H, dd, 8.4, 1.2) (1H, d, 15.0) 7.50 (1H, d, 15.6) 7.50 (1H, d, 15.6) 7.50 (1H, d, 15.6) (1H, d, 15.0) 6.46 (1H, d, 15.0) 6.31 (1H, d, 16.2) 6.42 (1H, d, 15.6) O Me 3.78 (3H, s) 3.79 (3H, s) dd, J = 2.4, 8.4 Hz, H-6 0 ), 7.51 (H, d, J = 2.4 Hz, H-2 0 ), 6.80 (1H, d, J = 8.4 Hz, H-5 0 ), 6.35 (1H, br-s, H-8), 6.16 (1H, br-s, H-6), 5.35 (1H, d, J = 7.8 Hz, H-1 00 ). 13 C NMR (DMSO-d 6, 150 MHz): d (C-4), (C-7), (C-5), (C-9), (C-2), (C- 4 0 ), (C-3 0 ), (C-3), (C-6 0 ), (C-1 0 ), (C- 5 0 ), (C-2 0 ), (C-10), (C-1 00 ), 98.8 (C-6), 93.6 (C-8), 75.8 (C-5 00 ), 73.2 (C-4 00 ), 71.2 (C-3 00 ), 67.9 (C-2 00 ), 60.1 (C-6 00 ). Quercetin-3-O-b-D-glucoside (peak 19): yellow powder, ESI- HRMS m/z: [M+H] + (calcd for C 21 H 21 O 12, ). 1 H NMR (DMSO-d 6, 600 MHz): d (1H, s, 5-OH), 7.60 (1H, dd, J = 2.4, 9.0 Hz, H-6 0 ), 7.51 (H, d, J = 2.4 Hz, H-2 0 ), 6.80 (1H, d, J = 9.0 Hz, H-5 0 ), 6.30 (1H, br-s, H-8), 6.10 (1H, br-s, H-6), 5.42 (1H, d, J = 7.2 Hz, H-1 00 ). 13 C NMR (DMSO-d 6, 150 MHz): d (C-4), (C-7), (C-5), (C-9), (C-2), (C-

6 1382 Q. Ren et al. / Food Chemistry 136 (2013) Table 2 13 C NMR spectral data for peak 33, 34, 35 and 36 in DMSO-d6. Fructose Peak 36 Peak 33 Peak 34 Peak Glucose Phenylpropanoids glu-6 0 feruloyl feruloyl feruloyl feruloyl O Me fruc-1 feruloyl p-coumaroyl feruloyl feruloyl O Me fruc-3 feruloyl p-coumaroyl p-coumaroyl p-coumaroyl O Me fruc-6 feruloyl p-coumaroyl p-coumaroyl feruloyl O Me ), (C-3 0 ), (C-3), (C-6 0 ), (C-1 0 ), (C-5 0 ), (C-2 0 ), (C-10), (C-1 00 ), 99.1 (C-6), 93.7 (C-8), 77.5 (C-5 00 ), 76.5 (C-3 00 ), 74.1 (C-2 00 ), 69.9 (C-4 00 ), 60.9 (C-6 00 ). Kaempferol-3-O-b-D-galactoside (peak 21): yellow powder, ESI- HRMS m/z: [M+H] + (calcd for C 21 H 21 O 11, ). 1 H NMR (DMSO-d 6, 600 MHz): d (1H, br-s, 5-OH), 8.05 (2H, d, J = 9.0 Hz, H-2 0, H-6 0 ), 6.84 (2H, d, J = 9.0 Hz, H-3 0, H-5 0 ), 6.38 (1H, br-s, H-8), 6.16 (1H, br-s, H-6), 5.37 (1H, d, J = 7.8 Hz, H-1 00 ). 13 C NMR (DMSO-d 6, 150 MHz): d (C-4), (C-7), (C- 5), (C-4 0 ), (C-2), (C-9), (C-3), (C-2 0, 6 0 ), (C-1 0 ), (C-3 0,5 0 ), (C-10), (C-1 00 ), 98.9 (C-6), 93.8 (C-8), 75.7 (C-5 00 ), 73.1 (C-4 00 ), 71.2 (C-3 00 ), 67.8 (C-2 00 ), 60.1 (C-6 00 ). Kaempferol-3-O-b-D-glucoside (peak 22): yellow powder, ESI- HRMS m/z: [M+H] + (calcd for C 21 H 21 O 11, ). 1 H NMR(DMSO-d 6, 600 MHz): d (1H, br-s, 5-OH), 8.02 (2H, d, J = 9.0 Hz, H-2 0, H-6 0 ), 6.87 (2H, d, J = 9.0 Hz, H-3 0, H-5 0 ), 6.38 (1H, br-s, H-8), 6.16 (1H, br-s, H-6), 5.44 (1H, d, J = 7.2 Hz, H-1 00 ). 13 C NMR (DMSO-d 6, 150 MHz): d (C-4), (C-7), (C-5), (C-4 0 ), (C-2), (C-9), (C-3), (C- 2 0, 6 0 ), (C-1 0 ), (C-3 0, 5 0 ), (C-10), 101.0(C-1 00 ), 98.9 (C-6), 93.7 (C-8), 77.5 (C-5 00 ), 76.4 (C-3 00 ), 74.2 (C-2 00 ), 69.9 (C-4 00 ), 60.8 (C-6 00 ). Quercetin-3-O-a-L-rhamnoside (peak 23): yellow powder, ESI- HRMS m/z: [M+H] + (calcd for C 21 H 21 O 11, ). 1 H NMR (DMSO-d 6, 600 MHz): d (1H, s, 5-OH), 7.28 (1H, d, J=2.4 Hz, H-2 0 ), 7.24 (1H, dd, J=2.4, 8.4 Hz, H-6 0 ), 6.84 (1H, d, J = 8.4 Hz, H-5 0 ), 6.35 (1H, br-s, H-8), 6.17(1H, br-s, H-6), 5.24 (1H, br-s, H-1 00 ), 0.80 (3H, d, J = 6.6 Hz, H rha CH 3 ). 13 C NMR (DMSO-d 6, 150 MHz): d (C-4), (C-7), (C-5), (C-9), (C-2), (C-4 0 ), (C-3 0 ), (C-3), (C-1 0 ), (C-6 0 ), (C-5 0 ), (C-2 0 ), (C-10), (C-1 00 ), 98.8 (C-6), 93.7 (C-8), 71.2 (C-4 00 ), 70.5 (C-3 00 ), 70.3 (C-2 00 ), 70.0 (C-5 00 ), 17.5 (C-6 00 ). Quercetin-3-O-[b-D-xyloxyl-(1? 2)-a-L-rhamnoside] (peak 24): yellow powder, ESI-HRMS m/z: [M+H] + (calcd for C 26 H 29 O 15, ). 1 H NMR (DMSO-d 6, 500 MHz): d (1H, s, 5-OH), 7.34 (1H, d, J=2.0 Hz, H-2 0 ), 7.25 (1H, dd, J=2.0, 8.0 Hz, H-6 0 ), 6.88 (1H, d, J = 8.0 Hz, H-5 0 ), 6.38 (1H, d, J=2.0 Hz, H-8), 6.19 (1H, d, J=2.0 Hz, H-6), 5.28 (1H, br-s, H-1 00 ), 4.14 (1H, d, J = 7.5 Hz, H ), 4.04 (1H, d, J = 2.0 Hz, H-2 00 ), 0.89 (3H, d, J = 6.5 Hz, H-rha-CH 3 ). 13 C NMR (DMSO-d 6, 125 MHz): d (C- 4), (C-7), (C-5), (C-9), (C-2), (C-4 0 ), (C-3 0 ), (C-3), (C-1 0 ), (C-6 0 ), (C-5 0 ), (C-2 0 ), (C ), (C-10), (C-1 00 ), 98.7 (C-6), 93.6 (C-8), 80.6 (C-2 00 ), 76.2 (C ), 73.7 (C ), 71.7 (C-4 00 ), 70.3 (C-3 00 ), 70.2 (C ), 69.3 (C-5 00 ), 65.7 (C ), 17.4 (C-6 00 ). N-trans-feruloyltyramine (peak 26): amorphous white powder, ESI-HRMS m/z: [M+H] + (calcd for C 18 H 21 O 4 N, ). 1 H NMR (DMSO-d 6, 600 MHz): d 9.40 (1H, br-s, -OH), d 9.16 (1H, br-s, -OH), 7.97 (1H, t, J=5.4 Hz, NH), 7.31 (1H, d, J = 15.6 Hz, H-3), 7.11(1H, d, J = 1.8 Hz, H-5), 7.01 (2H, d, J = 8.4 Hz, H-4 0, H-8 0 ), 6.98 (1H, dd, J = 8.4, 1.8 Hz, H-9), 6.79 (1H, d, J = 8.4 Hz, H-8), 6.68 (2H, d, J = 9.0 Hz, H-5 0, H-7 0 ), 6.43 (1H, d, J = 16.2 Hz, H-2), 3.80 (3H, s, 6-OCH 3 ), 3.32 (2H, m, H-1 0 ), 2.64 (2H, t, J = 7.2 Hz, H-2 0 ). 13 C NMR (DMSO-d 6, 150 MHz): d (C-1), (C-6 0 ), (C-7), (C-6), (C-3), (C- 3 0,4 0,8 0 ), (C-4), (C-9), (C-2), (C-8), (C-5 0,7 0 ), (C-5), 55.5 (-OCH 3 ), 40.6 (C-1 0 ), 34.4 (C-2 0 ) Analysis of reference and isolated compounds by HPLC-PDA/LTQ- FTICRMS The reference and isolated compounds were analyzed by HPLC- PDA/LTQ-FTICRMS. Their characteristic UV absorbance wavelength, accurate molecular weight and multiple-stage mass data were obtained in Table 3 and their fragmentation pathways were proposed Quinic acid derivatives. Chlorogenic acid (Peak 6, t R = min) displayed maximum UV absorption at 234 nm and 325 nm, [M+H] + ion at m/z 355. The MS 2 spectrum gave the base peak at m/z 163 [caffeic acid H 2 O+H] +, due to the loss of a quinic acid moiety (192 Da). In the MS 3 mass spectrum, ion at m/z 163 further fragmented to ion at m/z 145 [caffeic acid 2H 2 O+H] +, which was attributed to the loss of 18 Da. Then, fragment ion at m/z 117 [caffeic acid 2H 2 O CO + H] + was observed in the MS 4 mass spectrum, which resulted from the ion at m/z 145 for the loss of 28 Da.

7 Q. Ren et al. / Food Chemistry 136 (2013) Table 3 Identification of individual peaks from tartary buckwheat by FTICR-MS and ESI-MS n. Peak no. t R (min) Observed [M+H] + or [M+Na] + Calculated Error (ppm) k max (nm) ESI-MS n data (relative intensity, %) Identification and relative content (%) C 11 H 13 O 2 N ,262 MS 2 [205]: 188(100) Tryptophane b (0.02%) MS 3 [205? 188]: 146(100), 144(12) MS 4 [205? 188? 146]: 118(100) C 21 H 25 O ,278 MS 2 [453]: 435(11), 417(11), 301(68), (epi) Catechin-hexose b 291(100), 283(10), 273(17) (0.04%) MS 3 [453? 291]: 273(24), 165(44), 151(23), 139(100), 123(96) C 30 H 27 O MS 2 [579]: 427(100), 411(11), 409(67), Procyanidin B3 b 301(18), 291(46), 289(24), 247(23) (<0.01%) MS 3 [579? 427]: 409(100), 301(68), 287(18), 275(66), 247(12), 139(12) MS 3 [579? 409]: 299(16), 287(100), 283(26), 271(16), 259(34) C 30 H 27 O ,278 MS 2 [579]: 453(23), 439(10), 435(10), Procyanidin B1 b 427(100), 411(10), 409(55), 301(19), 289(14), (0.02%) 247(16) MS 3 [579? 427]: 409(100), 301(84), 287(12), 275(35), 247(11), 139(11) MS 3 [579? 409]: 391(13), 299(17), 287(100), 283(13), 259(60), 257(17), 245(10) C 15 H 15 O ,279 MS 2 [291]: 273(22), 165(49), 151(26), Catechin a (0.7%) 123(100) 139(97), MS 3 [291? 123]: 95(100) MS 3 [291? 139]: 111(100), 93(18), 67(19) C 16 H 19 O ,325 MS 2 [355]: 163(100) Chlorogenic acid a MS 3 [355? 163]: 145(100), 135(10) (4.75%) MS 4 [355? 163? 145]: 117(100) C 30 H 27 O ,278 MS 2 [579]: 427(89), 409(25), 301(10), Procyanidin B4 b (0.46%) 291(100), 289(89), 271(10) C 30 H 27 O ,278 MS 2 [579]: 427(100), 411(14), 409(50), Procyanidin B2 b (0.03%) 301(19), 291(50), 289(13), 247(20) MS 3 [579? 427]: 409(100), 301(79), 287(13) 275(50), 247(13), 139(10) MS 3 [579? 409]: 299(19), 287(100), 283(29), 271(13), 259(25) MS 4 [579? 427? 409]: 287(100) C 15 H 15 O ,279 MS 2 [291]: 273(21), 165(46), 151(27), Epicatechin a (0.04%) 139(97), 123(100) MS 3 [291? 123]: 95(100) MS 3 [291? 139]: 111(100), 93(10), 67(21) C 16 H 19 O ,285 MS 2 [339]: 147(100) Coumaroyl quinic acid b MS 3 [339? 147]: 119(100) (2.3%) C 17 H 21 O ,277 MS 2 [353]: 191(100) Methyl coumaroyl quinic MS 3 [353? 191]: 191(100), 176(11), 163(35) acid b (0.17%) C 17 H 21 O ,283 MS 2 [369]: 177(100) Feruloyl quinic acid b MS 3 [369? 177]: 145(100) (1.05%) MS 4 [369? 177? 145]: 117(100) C 45 H 37 O ,277 MS 2 [865]: 713(95), 695(45), 533(100), Procyanidin trimer 287(15) MS 3 [865? 533]: 515(25), 407(78), A-type b (0.20%) 287(100), 247(33) MS 3 [865? 713]: 695(100), 561(25), 543(42), 409(14), 287(19) MS 4 [856? 713? 695]: 677(12), 569(13), (continued on next page)

8 1384 Q. Ren et al. / Food Chemistry 136 (2013) Table 3 (continued) Peak no. t R (min) Observed [M+H] + or [M+Na] + Calculated Error (ppm) k max (nm) ESI-MS n data (relative intensity, %) Identification and relative content (%) 409(100), 287(25), 257(17) C 45 H 37 O ,277 MS 2 [865]: 713(100), 695(46), 575(10), Procyanidin trimer 533(98), 411(11), 287(16) A-type b (0.41%) MS 3 [865? 533]: 515(18), 407(64), 287(100), 247(29) MS 3 [865? 713]: 695(100), 561(23), 543(44), 427(11), 409(16), 287(26) MS 4 [856? 713? 695]: 569(19), 543(53), 409(100), 287(28), 257(10) C 22 H 19 O MS 2 [443]: 291(10), 273(100), 151(15), (epi) Catechin gallate b 139(11) MS 3 [433? 273]: 151(16), 147(15), (<0.01%) 123(100) C 27 H 31 O ,355 MS 2 [611]: 465(25), 303(100) Rutin a (0.02%) MS 3 [611? 303]: 303(13), 285(49), 274(12), 257(100), 247(35), 229(67), 165(53), 153(20), 137(14) MS 3 [611? 465]: 447(14), 303(100) MS 4 [465? 303]: 303(11), 285(64), 274(11), 257(100), 247(34), 229(25), 165(53),137(11) C 21 H 21 O ,354 MS 2 [465]: 303(100) Quercetin-3-O-b-Dgalactoside MS 3 [465? 303]: 303(14), 285(57), 274(13), 257(100), 247(32), 229(56), 165(44), 153(14), 137(15) a (1.41%) C 21 H 19 O ,340 MS 2 [479]: 303(100) Quercetin-O-glucuronide b MS 3 [479? 303]: 303(14), 285(60), (2.01%) 275(14), 274(15), 257(100), 247(24), 229(76), 165(51), 153(13), 137(20) MS 4 [479? 303? 257]: 229(100) C 21 H 21 O ,353 MS 2 [465]: 303(100) Quercetin-3-O-b-Dglucoside MS 3 [465? 303]: 303(10), 285(54), 275(11), 274(12), 257(100), 247(28), 229(64), 195(7), 165(53), 153(12), 137(15) MS 4 [465? 303? 257]: 229(100) a (5.99%) C 26 H 29 O ,348 MS 2 [581]: 449(23), 287(100) Kaempferol-O-pentosyl MS 3 [581? 287]: 287(28), 269(27), 259(12), 258(23), 241(100), 231(28), 213(55), 165(77), 153(30), 121(22), 111(10) hexoside b (1.21%) C 21 H 21 O ,348 MS 2 [449]: 287(100) Kaempferol-3-O-b-Dgalactoside MS 3 [449? 287]: 287(30), 269(37), 259(17), 258(31), 241(100), 231(41), 213 (73), 197(15), 185(11), 165(93), 153(38), 133(18) MS 4 [449? 287? 241]: 213(100) a (1.92%) C 21 H 21 O ,347 MS 2 [449]: 287(100) Kaempferol-3-O-b-Dglucoside MS 3 [449? 287]: 287(30), 269(37), 259(17), 258(31), 241(100), 231(41), 213 (73), 197(15), 185(10), 165(92), 153(38), 133(18) MS 4 [449? 287? 241]: 213(100) a (6.38%) C 21 H 21 O ,348 MS 2 [449]: 303(100) Quercetin-3-O-a-Lrhamnoside MS 3 [449? 303]: 303(17), 285(56), 274(11), 257(100), 247(34), 229(69), 165(46), 153(14), 137(17) MS 4 [449? 303? 257]: 229(100) a (5.66%)

9 Q. Ren et al. / Food Chemistry 136 (2013) Table 3 (continued) Peak no. t R (min) Observed [M+H] + or [M+Na] + Calculated Error (ppm) k max (nm) ESI-MS n data (relative intensity, %) Identification and relative content (%) C 26 H 29 O ,349 MS 2 [581]: 449(100), 431(10), 303(61) Quercetin-3-O-[b-D-xyloxyl- MS 3 [581? 303]: 303(44), 285(61), 274(13), 257(100), 247(33), 229(60), 165(53),153(15) MS 3 [581? 449]: 431(77), 413(100), 345(29), 303(11) MS 4 [581? 449? 413]: 395(100), 315(25) (1? 2)-a-L-rhamnoside] a (7.80%) C 20 H 19 O ,349 MS 2 [419]: 287(100) Kaempferol-O-pentoside b MS 3 [419? 287]: 287(43), 269(41), (0.51%) 241(100), 231(31), 213(66), 165(90), 153(44), 147(10), 133(22), 121(32), 111(12) C 18 H 21 O 4 N ,318 MS 2 [314]: 177(100) N-trans-feruloyltyramine a MS 3 [314? 177]: 145(100) (7.71%) MS 4 [314? 177? 145]: 117(100) MS 5 [314? 177? 145? 117]: 89(100) C 30 H 27 O MS 2 [611]: 593(11), 345(23), 309(80), 303(100), 291(28) MS 3 [611? 303]: 303(23), 285(67), 275(18), 257(100), 247(21), 229(66), 219(10), 213(11), 195(10), 165(43), 153(13), 137(14), 111(10) MS 3 [611? 309]: 147(100), 291(90) MS 4 [611? 309? 147]: 119(100) C 30 H 27 O MS 2 [611]: 593(10), 345(19), 309(84), 303(100), MS 3 [611? 303]: 303(40), 285(100, 275(30), 257(81), 247(34), 229(85), 213(30), 165(49), 153(28), 137(14), 111(12) MS 3 [611? 309]: 147(100), 291(10) Quercetin-O-coumaroyl hexose b (0.84%) Quercetin-O-coumaroyl hexose b (0.46%) C 30 H 27 O MS 2 [595]: 329(18), 309(75), 287(100) Kaempferol-O-coumaroyl MS 3 [595? 287]: 287(17), 269(44), 259(16), 258(26), 241(100), 231(37), 213(58), 203(10), 197(16), 185(15), 183(10), 165(93), 153(48), 137(11), 133(14), 121(24) MS 3 [595? 309]: 147(100) MS 4 [595? 309? 147]: 119(100) hexose b (1.06%) C 30 H 27 O ,328 MS 2 [595]: 309(69), 291(23), 287(100) Kaempferol-O-coumaroyl MS 3 [595? 287]: 287(41), 269(56), 259(13), 258(22), 243(22), 241(100), 231(20), 213(62), 197(16), 189(17), 165(74), 157(12), 153(30), 147(12), 133(19), 111(21) MS 3 [595? 309]: 291(93), 147(100) MS 4 [595? 309? 147]: 119(100) hexose b (2.23%) C 30 H 27 O MS 2 [595]: 309(10), 287(100), Kaempferol-O-coumaroyl MS 3 [595? 287]: 287(31), 269(28), 259(17), 258(46), 241(88), 231(35), 213(66), 185(16), 165(100), 153(56), 133(20), 121(28) MS 3 [595? 309]: 291(40), 147(100) MS 4 [595? 309? 147]: 119(100) hexose b (0.54%) C 39 H 40 O 17 Na ,312 MS 2 [803]: 641(100) 1, 3, 6-tri-p-coumaroyl MS 3 [803? 641]: 623(100), 477(56), 459(37), 405(43), 333(18), 331(23), 313(24) sucrose b (0.63%) C 49 H 48 O 20 Na ,314 MS 2 [979]: 641(100) 1, 3, 6-tri-p-coumaroyl feruloyl sucrose a MS 3 [979? 641]: 623(100), 477(50), (1.52%) 459(30), 405(36), 333(16), 331(23), 313(31) (continued on next page)

10 1386 Q. Ren et al. / Food Chemistry 136 (2013) Table 3 (continued) Peak no. t R (min) Observed [M+H] + or [M+Na] + Calculated Error (ppm) k max (nm) ESI-MS n data (relative intensity, %) Identification and relative content (%) C 50 H 50 O 21 Na ,317 MS 2 [1009]: 671(100) 3, 6-di-p-coumaroyl-1, 6 0 -diferuloyl sucrose a MS 3 [1009? 671]: 653(100), 489(24), (2.99%) 477(25), 459(6), 435(33), 363(12), 331(15), 313(22) C 51 H 52 O 22 Na ,322 MS 2 [1039]: 701(100) 1, 6, 6 0 -tri-feruloyl-3-pcoumaroyl sucrose a MS 3 [1039? 701]: 683(100), 519(20), (4.72%) 507(32), 489(11), 435(30), 363(17), 361(14), 343(17) C 52 H 54 O 23 Na ,327 MS 2 [1069]: 731(100) 1, 3, 6, 6 0 -tetra-feruoyl MS 3 [1069? 731]: 713(100), 537(28), 519(21), 465(27), 393(13), 361(10), 343(16) sucrose a (2.47%) UV spectra were not available due to low intensity. a indicates the compounds were unequivocally identified. b indicates the compounds were tentatively assigned. Fig. 3. Major mass fragmentations proposed for peak Flavan-3-ol derivatives. Catechin (peak 5, t R = min) and epicatechin (peak 9, t R = min) showed the same [M+H] + ion at m/z 291. Both of two peaks produced the typical fragments of m/z 273, 165, 151, 139 and 123 from ion at m/z 291 in the MS 2 spectrum, which arose from cleavage of the C ring. Then, in the MS 3 spectrum from the base peak ion at m/z 139 generated the characteristic fragment ions at m/z 111, 93 and 67. The fragment ion at m/z 123 produced the ion at m/z Flavonol derivatives. In tartary buckwheat extract, there are a great number of flavonol derivatives that are active chemical constituents associated with antioxidant and antidiabetic activities (Li et al., 2009). The major flavonol glycosides consist of two types of flavonol aglycones such as quercetin and kaempferol. UV spectrum and multi-stage mass data of flavonol aglycone and glycoside are valuable for characterization and identification. Peak 16, peak 17, peak 19, peak 23 and peak 24 belong to quercetin aglycone. Rutin (peak 16, t R = min) exhibited the protonated molecule [M+H] + ion at m/z 611. In the MS 2 spectrum, the fragment ion at m/z 465 derived from the loss of a rhamnose moiety [M 146] + and ion at m/z 303 with the highest intensity due to the loss of a glucose moiety [M ( )] + from the molecular ion. The MS 3 mass spectrum of m/z 303 presented the specific fragment ions at m/z 285, 257, 247, 229, 165 and 153, which characterized quercetin aglycone. It was established by comparison with previously reported data (Verardo et al., 2010). Quercetin-3-O-b-D-galactoside (peak 17, t R = min) and quercetin-3-o-b-d-glucoside (peak 19, t R = min) presented the same [M+H] + ion at m/z 465. In the MS 2 spectrum, the loss of a hexose moiety (162 Da) from the ion at m/z 465 gave the fragment ion at m/z 303 with the highest relative intensity. Then the similar fragmentation pattern of quercetin aglycone from the fragment ion at m/z 303 was observed in the MS 2 spectrum. Quercetin-3-O-a-L-rhamnoside (peak 23, t R = min) and quercetin-3-o-[b-d-d-xyloxyl-(1? 2)-a-L-rhamnoside] (peak 24, t R = min) illustrated [M+H] + ions at m/z 449 and at m/z 581, respectively. In the MS 2 spectrum of peak 23, the fragment ion at m/z 303 was attributed to the loss of a rhamnose moiety (146 Da) from m/z 449. Then the fragment pattern from the ion at m/z 303 was similar with those of peak 16, 17 and 19 in MS 3 spectrum. The [M+H] + ion at m/z 581 of peak 24 yielded the major product ion at m/z 449 with the highest relative intensity and ion at m/z 303 corresponding to the loss of a pentose (132 Da) moiety and

11 Q. Ren et al. / Food Chemistry 136 (2013) rhamnose (146 Da) moiety. The MS 3 mass spectrum of peak 24 from the fragment ion at m/z 303 was similar to that of quercetin aglycone. Kaempferol-3-O-b-D-galactoside (peak 21, t R = min) and kaempferol-3-o-b-d-glucoside (peak 22, t R = min) belonged to kaempferol aglycone and presented the same [M+H] + ions at m/ z 449. In the MS 2 spectrum, the prominent fragment ion at m/z 287 was assigned to the loss of a hexose moiety (162 Da) from [M+H] + ion at m/z 449. The MS 3 mass spectrum of m/z 287 illustrated the fragment ions at m/z 269, 241, 231, 213, 165 and 153, which were the characteristic fragmentations of kaempferol aglycone. In summary, two kinds of aglycones showed the difference maximum UV absorption, quercetin glycoside at nm and nm and kaempferol glycoside at nm and nm. Quercetin glycoside produced characteristic fragment ion at m/z 303, then further fragmented to characteristic ions at m/z 285, 257, 247, 229, 165 and 153, whereas kaempferol glycoside yielded diagnostic fragment ion at m/z 287, then gave rise to characteristic ions at m/z 269, 241, 231, 213, 165 and 153. The most commonly encountered sugar, followed by pentose (xylose and arabinose, 132 Da), deoxyhexose (rhamnose, 146 Da) and hexose (glucose and galactose, 162 Da). Two types of flavonol derivatives could be distinguished according to maximum UV absorption and fragmentation pattern Phenylpropanoid glycosides. Phenylpropanoid glycosides showed the characteristic maximum UV absorption wavelength at nm and nm. 1, 3, 6-tri-p-coumaroyl-6 0 -feruloyl sucrose (peak 33, t R = min) presented the [M+Na] + ion at m/z 979. The characteristic ion at m/z 641 with the highest relative intensity from [M+Na] + ion at m/z 979 was produced in the MS 2 spectrum, corresponding to the characteristic loss of feruloyl hexose moiety (338 Da). In the MS 3 spectrum, the four major fragments at m/z 623 [641 H 2 O] with the highest relative intensity, m/z 477 [641 H 2 O p-coumaroyl], m/z 459 [641 2H 2 O p-coumaroyl] and m/z 313 [641 2H 2 O 2 p-coumaroyl] were observed, due to the loss of H 2 O and p-coumaroyl moiety. The characteristic fragment ions indicated that one feruloyl moiety was located on the glucopyranose ring and three p-coumaroyl moieties were on the fructofuranose ring. Moreover, its structure was confirmed by NMR data and in agreement with the literature (Takasaki et al., 2001). 3, 6-di-p-coumaroyl-1, 6 0 -di-feruloyl sucrose (peak 34, t R = min) and 1, 6, 6 0 -tri-feruloyl-3-p-coumaroyl sucrose (peak 35, t R = min) showed [M+Na] + ions at m/z 1009 and 1039, respectively. Peak 35 had one more methoxyl group than peak 34. The characteristic ion at m/z 671 and 701 with the highest relative intensity from [M+Na] + ion at m/z 1009 and 1039 were produced in the MS 2 spectrum, corresponding to the characteristic loss of feruloyl hexose moiety (338 Da). The fragmentation pattern of peak 34 and peak 35 were similar to peak 33. 1, 3, 6, 6 0 -tetra-feruloyl sucrose (peak 36, t R = min) presented [M+Na] + ion at m/z The MS 2 fragmentations yielded ion at m/z 731 the highest relative intensity, corresponded to the loss of feruloyl hexose moiety (338 Da). In the MS 3 spectrum, the major fragments were m/z 713 [731 H 2 O], m/z 537 [731 H 2 O feruloyl], m/z 519 [731 2H 2 O feruloyl] and m/z 343 [731 2H 2 O 2 feruloyl], due to the loss of H 2 O and feruloyl moiety. The fragmentation scheme is given in Fig. 3. The typical fragmentation pathways for phenylpropanoid glycosides were the loss of feruloyl hexose moiety (338 Da), H 2 O, p-coumaroyl and feruloyl moiety subsequently Nitrogen compound. N-trans-feruloyltyramine (peak 26, t R = min) displayed the [M+H] + ion at m/z 314. The MS 2 spectrum showed the base peak at m/z 177, which was corresponded to feruloyl moiety. The MS 3 spectrum of ion m/z 177 produced the base peak at m/z 145, corresponding to the loss of (CH 3 OH, 32 Da) Tentative identification of tartary buckwheat by HPLC-PDA/LTQ- FTICRMS By comparing the information with the isolated compounds, the others were tentatively identified. Their structural information were collected and listed in Table Quinic acid derivatives. Peak 10, peak 11 and peak 12 showed similar fragmentation pattern to peak 6. Peak 10 (t R = min) exhibited [M+H] + ion at m/z , which corresponded to the molecular formula of C 16 H 19 O 8. In the MS 2 spectrum, ion at m/z 339 produced a base peak at m/z 147 [p-coumaric acid H 2 O+H] +, corresponding to the typical loss of quinic acid moiety (192 Da). The MS 3 spectrum ion at m/z 147 produced ion at m/z 119 [p-coumaric acid H 2 O CO + H] +, which was attributed to the loss of 28 Da. In contrast to literatures data (Alonso-Salces et al., 2004; Jaiswal & Kuhnert, 2011; Vallverdú-Queralt et al., 2011), peak 10 was tentatively identified as coumaroyl quinic acid. Peak 11 (t R = min) displayed [M+H] + ion at m/z , indicating the molecular formula of C 17 H 21 O 8. In the MS/MS spectrum, then it produced a base peak at m/z 191, corresponding to the loss of 162 Da [caffeic acid H 2 O]. The MS 3 spectrum of ion at m/z 191 yielded ions at m/z 176 and 163. Peak 11 was tentatively identified as methyl coumaroyl quinic acid (Rakesh & Nikolai, 2011). Peak 12 (t R = min) exhibited [M+H] + ion at m/z , corresponding to the molecular formula of C 17 H 21 O 9. In the MS/MS spectrum gave the major ion at m/z 177 [ferulic acid H 2 O+H] +, corresponding to the loss of quinic acid moiety (192 Da). The MS 3 spectrum of m/z 177 produced ion at m/z 145 [ferulic acid H 2 O CH 3 OH + H] +. The multi-stage mass data were in good agreement with the literatures (Jaiswal & Kuhnert, 2011; Vallverdú-Queralt et al., 2011). Peak 12 was tentatively identified as feruloyl quinic acid Flavan-3-ol derivatives. Peak 2 (t R = 9.81 min) showed maximum UV absorption wavelength at 232 nm and 278 nm, [M+H] + ion at m/z , corresponding to C 21 H 25 O 11. The MS 2 spectrum of ion at m/z 453 gave a base peak at m/z 291, corresponding to the loss of a hexose moiety (162 Da). The MS 3 mass spectrum of fragment ion at m/z 291 produced the characteristic fragments of m/z 273, 165, 151, 139 and 123, which were similar to those of (epi) catechin. Above all these data matched well with the literature (Verardo et al., 2010). Thus, peak 2 was tentatively identified as (epi) catechin-hexose. In nature the dimeric procyanidins exist as the B-type procyanidins, which consist of four major isomers such as B 1,B 2,B 3 and B 4 (Pekic, Kovac, Alonso, & Revilla, 1998; Sun & Miller, 2003). These isomers indicate the similar UV absorbance and same elemental composition. Multi-stage data demonstrates the similar fragmentation ions and relative intensities. However, the difference on retention time is likely to identify the procyanidins due to linkage or stereochemistry. Generally, the procyanidin B 2 on C 18 column is at the retention time between catechin and epicatechin, near to epicatechin (Verardo et al., 2010). In addition, the B-type procyanidins such as B 3,B 1,B 4 and B 2 were eluted sequence on C 18 column (Pekic et al., 1998; Sun & Miller, 2003). Peak 3 (t R = min), peak 4 (t R = min) and peak 7 (t R = min) exhibited [M+H] + ions at m/z (calcd for C 30 H 27 O 12 ), (calcd for C 30 H 27 O 12 ) and (calcd for C 30 H 27 O 12 ), respectively. The three peaks mass spectrum presented the specific fragments of m/z 427 and 409, which were the characteristic fragmentations in agreement with procyanidin B-type dimmers, by comparison with previously reported data