characterization and identification of the chemical...

Food Chemistry 136 (2013) 1377–1389

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Food Chemistry

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Characterization and identification of the chemical constituents from tartarybuckwheat (Fagopyrum tataricum Gaertn) by high performance liquidchromatography/photodiode array detector/linear ion trap FTICR hybrid massspectrometry

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 MedicalSciences, Beijing 100050, PR China

a r t i c l e i n f o

Article history:Received 3 June 2012Received in revised form 7 September 2012Accepted 14 September 2012Available online 23 September 2012

Keywords:Tartary buckwheatHPLC-PDA/LTQ-FTICRMSPhenlypropanoid glycosidesFlavonoids

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.09.052

⇑ Corresponding author. Tel.: +86 10 83154880; faxE-mail address: [email protected] (J. Zhang).

a b s t r a c t

In recent years tartary buckwheat has become popular healthful food due to its antioxidant, antidiabeticand antitumor activities. However, its chemical constituents have not yet been fully characterized andidentified. In this paper, a novel high performance liquid chromatography coupled with photodiode arraydetector and linear ion trap FTICR hybrid mass spectrometry (HPLC-PDA/LTQ-FTICRMS) method wasestablished to characterize and identify a total of 36 compounds by a single run. The retention time, max-imum UV absorption wavelength, accurate mass weight and characteristic fragment ions were collectedon line. To confirm the structures, 11 compounds were isolated and identified by MS and NMR experi-ments. 1, 3, 6, 60-tetra-feruloyl sucrose named taroside was a new phenlypropanoid glycoside, togetherwith 3, 6-di-p-coumaroyl-1, 60-di-feruloyl sucrose, 1, 6, 60-tri-feruloyl-3-p-coumaroyl sucrose, N-trans-feruloyltyramine and quercetin-3-O-[b-D-xyloxyl-(1 ? 2)-a-L-rhamnoside] were isolated for the firsttime from the Fagopyrum species. The research enriched the chemical information of tartary buckwheat.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Tartary buckwheat belongs to the Polygonaceae family, whichhas two main species, including common buckwheat (Fagopyrumesculentum Moench) and tartary buckwheat (Fagopyrum tataricumGaertn). Tartary buckwheat and common buckwheat originatedfrom the southwest China and the Himalayan hills (Ohnishi,1998). In recent years tartary buckwheat has gained much atten-tion, due to its benefits for human health. In food processing, tar-tary buckwheat has been used to make various healthful foodssuch as noodles, herb tea and crackers in Asian countries. Not onlytartary buckwheat leaves but also sprouts are consumed as nutri-tional vegetable. Therefore, tartary buckwheat is recognized as ahealthy food.

The pharmacological investigations demonstrated that tartarybuckwheat had a variety of pharmacological activities such as anti-oxidant activity, which was determined by 2, 2-diphenyl-1-picryl-hydrazyl (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 buckwheatprotein against human mammary cancer cell Bcap37 (Guo, Zhu,

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Zhang, & Yao, 2007) and antidiabetic activity of tartary buckwheatbran extract being investigated through male KK-Ay mice (type 2diabetic) and C57BL/6 mice (the control) (Yao et al., 2008). Liet al. reported that three flavonoids such as quercetin, isoquercetinand rutin from tartary buckwheat bran were effective inhibitorsagainst a-glucosidase (Li, Zhou, Gao, Bian, & Shan, 2009). In addi-tion, tartary buckwheat could reduce the level of total cholesterol,lower the concentration of blood glucose and regulate the lipidprofile (Wang et al., 2009; Yao et al., 2008).

Many phytochemical investigations mainly focused on commonbuckwheat. However, there were only a few systematic investiga-tions about tartary buckwheat. Previous studies on tartarybuckwheat revealed that some types of compounds had been iden-tified such as flavonoids (rutin, quercetin and quercitrin), C-glyco-sylflavones (orientin, isoorientin, vitexin and isovitexin) (Kim et al.,2007a,b; Kim et al., 2009), flavan-3-ol monomers (catechin andepicatechin) (Kim et al., 2009), organic acids (caffeic, ferulic, gallic,chlorogenic, (+)-osbeckic, 5-hydroxymethyl-2-furoic, proto-catechuic and p-hydroxybenzoic acids) (Kim et al., 2007a,b; Kimet 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 trans-reveratrol (Nemcová, Zima, Barek, & Janovská, 2011; Yang & Ren,2008) were reported on the occurrence of tartary buckwheat. It

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1378 Q. Ren et al. / Food Chemistry 136 (2013) 1377–1389

was just recently that seven phenylpropanoid glycosides includingtatarisides A-G had been reported in tartary buckwheat root(Zheng et al., 2012). Hence, it is necessary to extensively character-ize and identify the chemical constituents of tartary buckwheat.

High performance liquid chromatography coupled with massspectrometry plays an important role in the analysis for the com-plex and minor constituents of crude herb extract. Fourier trans-form ion cyclotron resonance mass spectrometry (FTICR-MS)provides excellent mass accuracy for the determination of elemen-tal composition and high mass resolution for the difficult chro-matographic separation. The LTQ MS can provide multi-stagemass analysis (MSn), which is automatically carried out by meansof data dependent scan experiments. The FTICR-MS collects fullscan MS data, then chooses the most intense parent ion or theparent ions of interest for multi-stage mass fragmentation. Thetarget ions are fragmented. The cycle is repeated throughout theduration of the acquisition and provides a great deal of data, whichis best used for structural elucidation (Vallverdú-Queralt, Jáuregui,Di Lecce, Andrés-Lacueva, & Lamuela-Raventós, 2011).

The objective of current study is to develop a comprehensiveanalysis method for the chemical profile of tartary buckwheat andidentify the major and minor constituents by HPLC-PDA/LTQ-FTICRMS. In this paper, accurate mass and multiple-stage mass datawere collected on line. Then the structures of major constituentswere confirmed by spectroscopic methods and their characteristicfragmentation patterns were summarized. The minor chemicalconstituents were identified on the basis of their accurate massand characteristic fragmentation behavior of known compounds.

2. Materials and methods

2.1. Materials

Chlorogenic acid, rutin, catechin, and epicatechin were purchasedfrom the National Institute for Control of Pharmaceutical and Biolog-ical Products (Beijing, China). Quercetin-3-O-b-D-glucoside (peak 19),kaempferol-3-O-b-D-galactoside (peak 21), kaempferol-3-O-b-D-glu-coside (peak 22), quercetin-3-O-b-D-galactoside (peak 17), querce-tin-3-O-a-L-rhamnoside (peak 23), N-trans-feruloyltyramine (peak26), quercetin-3-O-[b-D-xyloxyl-(1 ? 2)-a-L-rhamnoside] (peak 24),1, 3, 6-tri-p-coumaroyl-60-feruloyl sucrose (peak 33), 3, 6-di-p-cou-maroyl-1, 60-di-feruloyl sucrose (peak 34), 1, 6, 60-tri-feruloyl-3-p-coumaroyl sucrose (peak 35), 1, 3, 6, 60-tetra-feruloyl sucrose (peak36) were isolated from tartary buckwheat in our laboratory. The pur-ity of reference substances were determined to be higher than 98% byHPLC. These structures were confirmed by their MS, 1D NMR and 2DNMR. The structures are shown in Fig. 1.

The total plant of tartary buckwheat (F. tataricum Gaertn) withroot, stem, and leaf was collected in the wild from HuiZe, Yunnanprovince by Professor Lin Ma during in July 2010. The collectedplant material was dried in the shade. The voucher specimen(No.201008) was deposited at the Institute of Materia Medica, Chi-nese Academy of Medical Sciences, China.

Acetonitrile of LC/MS reagent grade was supplied by Mallinck-rodt Baker, Inc. (Phillipsburg, NJ, USA). Deionized water was puri-fied by Millipore water purification system (Millipore, MA, USA).Analytical grade dichlormethane, methanol and acetic acid werepurchased from Beijing Chemical Corporation (Beijing, China).Sephadex LH-20 was obtained from GE Healthcare (made in Swe-den). Silica gel (100–200 mesh) was supplied by Qingdao MarineChemical Company (Qingdao, People’s Republic of China).

2.2. Sample preparation

The dry tartary buckwheat was cut into small pieces. Accurately1.0 g of tartary buckwheat was weighed and transferred into a

glass scintillation vial with 5 mL of 70% aqueous ethanol, whichwas treated with ultrasonication for 30 min at ambienttemperature. The extract was centrifuged at 4500 rpm for10 min, then filtered through a 0.22 lm filter and analyzed byHPLC-PDA/LTQ-FTICRMS.

2.3. Optimization of extract methods

To achieve good extraction efficiency, sample preparation wasoptimized. Two kinds of routine extract methods such as ultrason-ication and reflux were investigated. Ultrasonication (100 W,40 kHz) was in 5 mL different extraction solvents including meth-anol, 70% aqueous methanol, ethanol and 70% aqueous ethanolwith extraction time 30 min at ambient temperature. Refluxextraction in a water bath (45 �C) for 30 min with 5 mL four kindsof different solvents. Ultrasonic extraction was selected, because itwas more simple and faster than reflux. Then, various extractionsolvents including methanol, 70% aqueous methanol, ethanol and70% aqueous ethanol were tried. 70% aqueous ethanol could ex-tract a wide variety of compounds, which was of great benefit toprofile the chemical constituents of tartary buckwheat.

2.4. HPLC-PDA/LTQ-FTICRMS analysis

HPLC-PDA analysis was performed on a Finnigan Surveyor LCplus system (Thermo Fisher, Co. Ltd., San Jose, CA USA), equippedwith a surveyor MS pump plus and surveyor autosampler. Chro-matographic separation was carried out on a Kinetex C18 reversephase column (4.6 � 100 mm, 2.6 lm, Phenomenex). PDA detectorrecorded from 200 to 400 nm. The detection monitored simulta-neously at 210 nm, 280 nm and 320 nm. The mobile phaseconsisted of water containing 0.1% acetic acid (v/v) (A) and aceto-nitrile (B). Gradient elution was applied as follows: 0–20 minfor 6–16% B; 20–38 min for 16–20% B; 38–48 min for 20–33% B;48–68 min for 33–38% B; 68–70 min for 38–100% B; 70–75 minfor 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 temperaturewas maintained at 35 �C. The injection volume was 2 lL.

The FTICR-MS full scan and multi-stage mass experiments werecarried out on Finnigan LTQ FT (Thermo Fisher, Co. Ltd., San Jose,CA USA), which contained two kinds of gas that ultra high purityhelium was used as collision gas and high purity nitrogen as neb-ulizer gas. The optimized electrospray ion source parameters wereas follows: ion spray voltage at 3.5 kV, capillary temperature at250 �C, capillary voltage at 30 V, sheath gas flow rate at 45 (arbi-trary units), auxiliary gas flow rate at 10 (arbitrary units), sweepgas flow rate at 3 (arbitrary units) and tube lens offset voltage at90 V. Positive ionization gained a better result than negative ioni-zation. Most of the peaks were detected, which signals corre-sponded to the protonated ion [M+H]+ or the sodium adduct ion[M+Na]+. The effluent from HPLC system was split (1:1) andsprayed into mass spectrometer with an electrospray interfaceoperating in positive ionization for full scan in a m/z range of100–1800, with 3 microscans and a maximum ion injection timeof 200 ms. The data was acquired and analyzed by means of Xcal-ibur 2.0 software (Thermo Fisher Scientific, Waltham, MA).

2.5. 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 undervacuum 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 parti-tioned extracts by TLC and HPLC-MS, the EtOAc extract (20 g)

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-D-galactoside, (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-60-feruloyl sucrose, (9) 3, 6-di-p-coumaroyl-1,60-di-feruloyl sucrose, (10) 1, 6, 60-tri-feruloyl-3-p-coumaroyl sucrose, (11) 1, 3, 6, 60-tetra-feruloylsucrose.

Q. Ren et al. / Food Chemistry 136 (2013) 1377–1389 1379

enriched the target components such as flavonoids and phenyl-propanoid glycosides and was subjected to 200–300 mesh silicagel column (60 � 1000 mm i.d.) eluting with CH2Cl2 and MeOH(99:1 ? 0:100), and then gained eight fractions (A–H). The all frac-tions were monitored by TLC and HPLC-MS. The fraction D, E, F, Gand H concentrated the target compounds were pooled for furtherisolation, and the fraction A–C were laid aside for later studies.

Fraction D (1.8 g) was further applied to Sephadex LH-20 col-umn (50 � 800 mm i.d.) and eluted with CH2Cl2 and MeOH (1:1)to yield 60 sub-fractions. The sub-fractions from 21 to 43 werepooled and concentrated, then subjected to preparative HPLC ZOR-BAX Eclipse XDB-C18 (9.4 � 250 mm, 5 lm) column. The columnwas eluted by CH3CN and H2O containing 5% MeOH (19:81) withflow 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(200–300 mesh) column (50 � 800 mm i.d.) and eluted with CH2-

Cl2 and MeOH (9:1) to yield 30 sub-fractions. Sub-fractions from6 to 21 (2.35 g) were pooled and concentrated, then successivelyseparated on Sephadex LH-20 column for further purification withCH2Cl2-MeOH (1:1) and gained 55 sub-fractions, and then usedZORBAX Eclipse XDB-C18 (9.4 � 250 mm, 5 lm) column with theflow rate at 4.0 mL/min, detection at 320 nm, eluting with CH3CNand H2O containing 5% MeOH (32:68), which yielded peak 33(10.5 mg) from sub-fractions 46 to 50, peak 34 (11.2 mg) fromsub-fractions 41 to 43, peak 35 (15.9 mg) from sub-fractions 34to 38 and peak 36 (14.8 mg) from sub-fractions 22 to 33.

Fraction F (2.1 g) was chromatographed on Sephadex LH-20 col-umn (50 � 800 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-C18

(9.4 � 250 mm, 5 lm) column, a flow rate of 4.0 mL/min, detectionat 320 nm, eluting with CH3CN and H2O containing 5% MeOH(15:85).

Fraction G (2.0 g) was separated by Sephadex LH-20 column(50 � 800 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 C18 (10 � 250 mm, 10 lm)column with the flow rate at 4.0 mL/min, detection at 320 nm,eluting with CH3CN and H2O containing 5% MeOH (15:85), thenpeak 19 (7.1 mg), peak 21 (6.6 mg) and peak 22 (8.3 mg) were ob-tained. Sub-fractions from 37 to 48 were pooled and gained peak17 (7.7 mg).

Fraction H (1.2 g) was carried out on Sephadex LH-20 column(50 � 800 mm i.d.) by elution with MeOH to yield 56 sub-fractions.Sub-fractions from 25 to 33 were pooled and further purified bypreparative HPLC Waters Xbridge Prep C18 (10 � 250 mm, 10 lm)column with flow rate at 3.0 mL/min, detection at 320 nm, elutingwith CH3CN and H2O containing 5% MeOH (19:81), then gainedpeak 24 (18.6 mg).

2.6. NMR spectroscopy analysis

NMR spectra were obtained on a VNS-600 MHz spectrometeroperating at a proton NMR frequency of 599.693 MHz and carbonNMR frequency of 149.923 MHz. Samples were added to a 5 mmNMR tube with 450 lL DMSO-d6. Chemical shifts were given in d(ppm) scale relative to solvent peaks as reference. Acquisitionparameters were as follows: spectral width 6000 Hz, relaxation de-lay 1.0 s, number of scans 3000, pulse width 45�, and acquisitiontime 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 spec-trometry and PDA detector are capable of providing accurate mass,elemental composition, multiple-stage mass data and maximumUV absorption wavelength for characterization and identificationof tartary buckwheat.

The isolated reference substances from tartary buckwheat wereanalyzed by HPLC-PDA/LTQ-FTICRMS. The useful information such


as characteristic fragment ions and fragmentation pattern weresummarized. Unknown compounds were identified by comparingwith the information of reference substances. Moreover, the frag-mentation patterns of known compounds were utilized in correla-tion to propose the structures of unknown compounds.

3.2. Characteristic HPLC-PDA/LTQ-FTICRMS profile of tartarybuckwheat

3.2.1. Optimization of the chromatographic conditionsTo achieve good separation and profile comprehensive constit-

uents, the column, mobile phase and elution program were inves-tigated. The different types of columns such as Restek pinnacle IIC18 column (4.6 � 250 mm, 5 lm), Shiseido Capcell PAK CR type(1:4) and type (1:50) columns (4.6 � 250 mm, 5 lm), ZORBAX ser-ies including Extend C18 column, XDB C18 column, Eclipse Plus C18

column, SB-Phenyl column (4.6 � 250 mm, 5 lm) and Kinetex C18

column (4.6 � 100 mm, 2.6 lm, Phenomenex) were investigated.Phenlypropanoid glycosides (peak 33, 34, 35 and 36) were difficultto separate. The ZORBAX SB-Phenyl column could provide wellseparation for them, but poor separation for flavonoids. The Kine-tex C18 reverse phase column exhibited good chromatographic sep-aration for the most constituents and was chosen for analysis. Themobile phase of acetonitrile and water consisting 0.1% acetic acidprovided much better resolution for individual peaks. Due to thedifference in polarity between phenlypropanoid glycosides andflavonoids, gradient elution was adapted to probe as many constit-uents as possible in a single run.

3.2.2. HPLC-PDA/LTQ-FTICRMS profile of tartary buckwheatThe extract of tartary buckwheat was analyzed by HPLC-PDA/

LTQ-FTICRMS and the chemical profile was achieved. The represen-tative UV-absorption chromatogram (A), total ion chromatogram(B) and five extract ion chromatograms including quinic acid deriv-atives (C), flavan-3-ol derivatives (D), flavonol derivatives (E), phe-nylpropanoid glycosides (F), and nitrogen compounds (G) wereobtained in Fig. 2.

3.3. Identification of isolated compounds from tartary buckwheat

In order to probe the constituents of tartary buckwheat on thebase of their characteristic UV absorbance wavelength and mass

Fig. 2. The representative UV-absorption chromatogram (A), total ion chromatogram (B)derivatives (D), flavonol derivatives (E), phenylpropanoid glycosides (F), and nitrogen co

data, the major chemical constituents were isolated as referencesubstances and identified by HRMS and NMR data.

3.3.1. Structural determination of isolated compounds by NMRPeak 36 was a new compound, which was obtained as a white

powder. Its molecular formula was determined to be C52H54O23

by ESI-HRMS (m/z 1069.29431 [M+Na]+, calculated for (calcd for)C52H54O23Na).

The NMR data (Table 1) suggested the existence of four feruloylgroups and a sucrose moiety. Furthermore, the 1H NMR spectrum ofpeak 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); d6.69 (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) and7.26 (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 methox-yls at d 3.76 (3H, s), 3.74 (3H, s), 3.74 (3H, s), 3.78 (3H, s) were exhib-ited in the 1H NMR spectrum. The 1H and 13C NMR resonance valueswere similar to the reported literature (Takasaki, Kuroki, Kozuka, &Konoshima, 2001) for 1, 6, 60-triferuloyl-3-p-coumaroyl sucrose.

In the HMBC spectrum, phenyl protons correlated to olefinic car-bons, in addition olefinic proton correlated to each ester carbonylcarbon, such as correlations from H-60 of glucose to C-90 0 carbonyl,and from H-1, H-3, and H-6 of fructose to C-90 0 0, C-90 0 0 0, and C-90 0 0 0 0

carbonyls, which confirmed the linkage positions. The completeassignment of protons and carbons were established by analysesof 1H–1H COSY, HSQC, TOCSY and HMBC spectrum. On the basisof the spectral data, peak 36 was confirmed to be 1, 3, 6, 60-tetra-feruloyl sucrose, named taroside.

NMR spectroscopic data for peak 36, peak 33, peak 34 and peak35 are shown in Table 1 and Table 2. By comparing the literaturedata (Takasaki et al., 2001), peak 33, peak 34 and peak 35 wereidentified as 1, 3, 6-tri-p-coumaroyl-60-feruloyl sucrose, 3, 6-di-p-coumaroyl-1, 60-di-feruloyl sucrose and 1, 6, 60-tri-feruloyl-3-p-coumaroyl sucrose.

Quercetin-3-O-b-D-galactoside (peak 17): yellow powder, ESI-HRMS m/z: 465.10287 [M+H]+ (calcd for C21H21O12, 465.10275).1H NMR (DMSO-d6, 600 MHz): d 12.60 (1H, br-s, 5-OH), 7.65 (1H,

and five extract ion chromatograms including quinic acid derivatives (C), flavan-3-olmpounds (G).

Table 11H NMR spectral data for peak 33, 34, 35 and 36 in DMSO-d6.

Fructose Peak 36 Peak 33 Peak 34 Peak 35

1 4.10 (2H, m) 4.06 (2H, m) 4.09 (2H, m) 4.10 (2H, m)23 5.44 (1H, d, 9.0) 5.43 (1H, d, 8.4) 5.44 (1H, d, 9.0) 5.44 (1H, d, 9.0)4 4.38 (1H, m) 4.38 (1H, m) 4.39 (1H, m) 4.37 (1H, m)5 4.03 (1H, m) 4.08 (1H, m) 4.07 (1H, m) 4.03 (1H, m)6 4.33 (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)

Glucose10 5.22 (1H, d, 3.6) 5.21 (1H, d, 3.6) 5.23 (1H, d, 3.0) 5.22 (1H, d, 3.6)20 3.25 (1H, m) 3.26 (1H, m) 3.25 (1H, m) 3.27 (1H, m)30 3.41 (1H, m) 3.41 (1H, m) 3.41 (1H, m) 3.41 (1H, m)40 3.11 (1H, m) 3.11 (1H, m) 3.11 (1H, m) 3.12 (1H, m)50 4.06 (1H, m) 4.02 (1H, m) 4.02 (1H, m) 4.05 (1H, m)60 4.08 (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)20-OH 4.90 (1H, d, 6.0) 4.87 (1H, br) 4.88 (1H, br) 4.88 (1H, br)30-OH 4.88 (1H, d, 4.8) 4.87 (1H, br) 4.88 (1H, br) 4.88 (1H, br)40-OH 5.33 (1H, d, 5.4) 5.30 (1H, br) 5.33 (1H, d, 4.8) 5.34 (1H, br)

Phenylpropanoidsglu-60 feruloyl feruloyl feruloyl feruloyl10 0

20 0 7.22 (1H, d, 1.2) 7.23 (1H, d, 1.2) 7.22 (1H, d, 1.8) 7.22 (1H, d, 1.2)30 0

40 0

50 0 6.69 (1H, d, 8.4) 6.69 (1H, d, 7.8) 6.71 (1H, d, 8.4) 6.71 (1H, d, 7.8)60 0 7.01 (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)70 0 7.49 (1H, d, 15.2) 7.45 (1H, d, 15.0) 7.50 (1H, d, 15.6) 7.45 (1H, d, 15.6)80 0 6.46 (1H, d, 15.0) 6.30 (1H, d, 16.2) 6.45 (1H, d, 16.2) 6.45 (1H, d, 15.6)90 0

O–Me 3.76 (3H, s) 3.74 (3H, s) 3.79 (3H, s) 3.74 (3H, s)fruc-1 feruloyl p-coumaroyl feruloyl feruloyl10 0 0

20 0 0 7.22 (1H, d, 1.2) 7.50 (1H, d, 8.4) 7.22 (1H, d, 1.8) 7.22 (1H, d, 1.2)30 0 0 6.68–6.7440 0 0

50 0 0 6.69 (1H, d, 7.8) 6.68–6.74 6.73 (1H, d, 7.8) 6.73 (1H, d, 7.8)60 0 0 6.98 (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)70 0 0 7.51 (1H, d, 15.6) 7.60 (1H, d, 15.6) 7.58 (1H, d, 15.0) 7.52 (1H, d, 16.2)80 0 0 6.44 (1H, d, 15.6) 6.36 (1H. d, 16.2) 6.36 (1H, d, 16.2) 6.42 (1H, d, 15.6)90 0 0

O–Me 3.74 (3H, s) 3.74 (3H, s) 3.79 (3H, s)fruc-3 feruloyl p-coumaroyl p-coumaroyl p-coumaroyl10 0 0 0

20 0 0 0 7.26 (1H, d, 1.2) 7.47 (1H, d, 8.4) 7.40 (1H, d, 8.4) 7.47 (1H, d, 8.4)30 0 0 0 6.68–6.74 6.70 (1H, d, 8.4) 6.70 (1H, d, 9.0)40 0 0 0

50 0 0 0 6.73 (1H, d, 7.8) 6.68–6.74 6.70 (1H, d, 8.4) 6.70 (1H, d, 9.0)60 0 0 0 7.09 (1H, dd, 7.8, 1.2) 7.47 (1H, d, 8.4) 7.40 (1H, d, 8.4) 7.47 (1H, d, 8.4)70 0 0 0 7.57 (1H, d, 15.6) 7.51 (1H, d, 15.0) 7.51 (1H, d, 15.6) 7.58 (1H, d, 15.6)80 0 0 0 6.42 (1H, d, 15.0) 6.35 (1H, d, 16.2) 6.42 (1H, d, 16.2) 6.36 (1H, d, 16.2)90 0 0 0

O–Me 3.74 (3H, s)fruc-6 feruloyl p-coumaroyl p-coumaroyl feruloyl10 0 0 0 0

20 0 0 0 0 7.23 (1H, d, 1.2) 7.40 (1H, d, 9.0) 7.47 (1H, d, 9.0) 7.24 (1H, d, 1.2)30 0 0 0 0 6.68 (1H, d, 8.4) 6.69 (1H, d, 8.4)40 0 0 0 0

50 0 0 0 0 6.71 (1H, d, 8.4) 6.68 (1H, d, 8.4) 6.69 (1H, d, 8.4) 6.71 (1H, d, 8.4)60 0 0 0 0 7.04 (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)70 0 0 0 0 7.45 (1H, d, 15.0) 7.50 (1H, d, 15.6) 7.50 (1H, d, 15.6) 7.50 (1H, d, 15.6)80 0 0 0 0 6.42 (1H, d, 15.0) 6.46 (1H, d, 15.0) 6.31 (1H, d, 16.2) 6.42 (1H, d, 15.6)90 0 0 0 0

O–Me 3.78 (3H, s) 3.79 (3H, s)


dd, J = 2.4, 8.4 Hz, H-60), 7.51 (H, d, J = 2.4 Hz, H-20), 6.80 (1H, d,J = 8.4 Hz, H-50), 6.35 (1H, br-s, H-8), 6.16 (1H, br-s, H-6), 5.35(1H, d, J = 7.8 Hz, H-10 0). 13C NMR (DMSO-d6, 150 MHz): d 177.3(C-4), 164.4 (C-7), 161.2 (C-5), 156.3 (C-9), 156.1 (C-2), 148.5 (C-40), 144.8 (C-30), 133.4 (C-3), 121.9 (C-60), 121.0 (C-10), 115.8 (C-50), 115.1 (C-20), 103.6 (C-10), 101.8 (C-10 0), 98.8 (C-6), 93.6 (C-8),75.8 (C-50 0), 73.2 (C-40 0), 71.2 (C-30 0), 67.9 (C-20 0), 60.1 (C-60 0).

Quercetin-3-O-b-D-glucoside (peak 19): yellow powder, ESI-HRMS m/z: 465.10251 [M+H]+ (calcd for C21H21O12, 465.10275).1H NMR (DMSO-d6, 600 MHz): d 12.60 (1H, s, 5-OH), 7.60 (1H,dd, J = 2.4, 9.0 Hz, H-60), 7.51 (H, d, J = 2.4 Hz, H-20), 6.80 (1H, d,J = 9.0 Hz, H-50), 6.30 (1H, br-s, H-8), 6.10 (1H, br-s, H-6), 5.42(1H, d, J = 7.2 Hz, H-10 0). 13C NMR (DMSO-d6, 150 MHz): d 177.1(C-4), 164.1 (C-7), 161.1 (C-5), 156.4 (C-9), 155.8 (C-2), 148.4 (C-

Table 213C NMR spectral data for peak 33, 34, 35 and 36 in DMSO-d6.

Fructose Peak 36 Peak 33 Peak 34 Peak 35

1 63.5 63.8 63.9 63.82 101.3 101.5 101.5 101.53 76.0 76.3 76.3 76.34 71.8 72.1 72.1 72.05 79.3 79.3 79.3 79.46 64.7 64.9 64.9 64.9

Glucose10 91.4 91.6 91.5 91.620 70.7 70.9 70.9 70.930 72.5 72.8 72.8 72.840 70.0 70.3 70.2 70.350 70.7 70.9 70.9 70.960 64.1 64.3 64.4 64.3

Phenylpropanoidsglu-60 feruloyl feruloyl feruloyl feruloyl10 0 125.4 125.5 125.6 125.620 0 110.8 110.9 110.9 111.530 0 147.7 147.9 147.9 147.940 0 149.1 149.4 149.4 149.450 0 115.2 115.4 115.4 115.460 0 123.1 123.3 123.2 123.370 0 145.0 145.1 145.2 145.580 0 114.0 113.7 114.2 113.990 0 166.5 166.5 166.7 166.7O–Me 55.3–55.5 55.5 55.7 55.5–55.7fruc-1 feruloyl p-coumaroyl feruloyl feruloyl10 0 0 125.2 124.9 125.0 125.520 0 0 110.8 130.5 111.5 110.930 0 0 147.7 115.8 147.9 147.940 0 0 149.2 160.0 149.5 149.650 0 0 115.2 115.8 115.4 115.660 0 0 123.3 130.5 123.1 123.170 0 0 145.3 145.1 146.2 146.280 0 0 113.8 113.2 113.6 113.890 0 0 165.8 165.9 165.9 165.9O–Me 55.3–55.5 55.5 55.5–55.7fruc-3 feruloyl p-coumaroyl p-coumaroyl p-coumaroyl10 0 0 0 125.2 124.9 125.4 125.020 0 0 0 111.3 130.4 130.4 130.430 0 0 0 147.7 115.8 115.7 115.840 0 0 0 149.3 160.0 159.9 159.950 0 0 0 115.4 115.8 115.7 115.860 0 0 0 122.9 130.4 130.4 130.470 0 0 0 146.0 145.1 145.2 145.180 0 0 0 113.4 113.6 113.8 113.690 0 0 0 165.7 165.9 165.9 165.9O–Me 55.3–55.5fruc-6 feruloyl p-coumaroyl p-coumaroyl feruloyl10 0 0 0 0 125.3 124.9 125.0 125.420 0 0 0 0 110.7 130.3 130.3 111.030 0 0 0 0 147.7 115.7 115.7 147.940 0 0 0 0 149.3 160.1 159.9 149.450 0 0 0 0 115.2 115.7 115.7 115.460 0 0 0 0 123.0 130.3 130.3 123.270 0 0 0 0 145.3 145.9 145.1 145.280 0 0 0 0 113.7 114.2 113.6 114.290 0 0 0 0 166.3 166.7 166.5 166.5O–Me 55.3–55.5 55.5–55.7


40), 144.9 (C-30), 133.1 (C-3), 121.5 (C-60), 120.9 (C-10), 115.9 (C-50),115.1 (C-20), 103.3 (C-10), 101.0 (C-10 0), 99.1 (C-6), 93.7 (C-8), 77.5(C-50 0), 76.5 (C-30 0), 74.1 (C-20 0), 69.9 (C-40 0), 60.9 (C-60 0).

Kaempferol-3-O-b-D-galactoside (peak 21): yellow powder, ESI-HRMS m/z: 449.10751 [M+H]+ (calcd for C21H21O11, 449.10784). 1HNMR (DMSO-d6, 600 MHz): d 12.58 (1H, br-s, 5-OH), 8.05 (2H, d,J = 9.0 Hz, H-20, H-60), 6.84 (2H, d, J = 9.0 Hz, H-30, H-50), 6.38 (1H,br-s, H-8), 6.16 (1H, br-s, H-6), 5.37 (1H, d, J = 7.8 Hz, H-10 0). 13CNMR (DMSO-d6, 150 MHz): d 177.3 (C-4), 164.3 (C-7), 161.1 (C-5), 159.9 (C-40), 156.4 (C-2), 156.1 (C-9), 133.1 (C-3), 130.9 (C-20,60), 120.9 (C-10), 115.0 (C-30, 50), 103.5 (C-10), 101.7 (C-10 0), 98.9(C-6), 93.8 (C-8), 75.7 (C-50 0), 73.1 (C-40 0), 71.2 (C-30 0), 67.8 (C-20 0),60.1 (C-60 0).

Kaempferol-3-O-b-D-glucoside (peak 22): yellow powder, ESI-HRMS m/z: 449.10764 [M+H]+ (calcd for C21H21O11, 449.10784).1H NMR(DMSO-d6, 600 MHz): d 12.59 (1H, br-s, 5-OH), 8.02 (2H,d, J = 9.0 Hz, H-20, H-60), 6.87 (2H, d, J = 9.0 Hz, H-30, H-50), 6.38(1H, br-s, H-8), 6.16 (1H, br-s, H-6), 5.44 (1H, d, J = 7.2 Hz, H-10 0).13C NMR (DMSO-d6, 150 MHz): d 177.3 (C-4), 164.3 (C-7), 161.2(C-5), 159.9 (C-40), 156.4 (C-2), 156.0 (C-9), 133.1 (C-3), 130.8 (C-20, 60), 120.9 (C-10), 115.1 (C-30, 50), 103.6 (C-10), 101.0(C-10 0),98.9 (C-6), 93.7 (C-8), 77.5 (C-50 0), 76.4 (C-30 0), 74.2 (C-20 0), 69.9(C-40 0), 60.8 (C-60 0).

Quercetin-3-O-a-L-rhamnoside (peak 23): yellow powder, ESI-HRMS m/z: 449.10785 [M+H]+ (calcd for C21H21O11, 449.10784).1H NMR (DMSO-d6, 600 MHz): d 12.64 (1H, s, 5-OH), 7.28 (1H, d,J = 2.4 Hz, H-20), 7.24 (1H, dd, J = 2.4, 8.4 Hz, H-60), 6.84 (1H, d,J = 8.4 Hz, H-50), 6.35 (1H, br-s, H-8), 6.17(1H, br-s, H-6), 5.24(1H, br-s, H-10 0), 0.80 (3H, d, J = 6.6 Hz, H–rha–CH3). 13C NMR(DMSO-d6, 150 MHz): d 177.6 (C-4), 164.5 (C-7), 161.2 (C-5),157.1 (C-9), 156.5 (C-2), 148.5 (C-40), 145.2 (C-30), 134.1 (C-3),121.1 (C-10), 120.7 (C-60), 115.6 (C-50), 115.4 (C-20), 103.8 (C-10),101.8 (C-10 0), 98.8 (C-6), 93.7 (C-8), 71.2 (C-40 0), 70.5 (C-30 0), 70.3(C-20 0), 70.0 (C-50 0), 17.5 (C-60 0).

Quercetin-3-O-[b-D-xyloxyl-(1 ? 2)-a-L-rhamnoside] (peak24): yellow powder, ESI-HRMS m/z: 581.14990 [M+H]+ (calcd forC26H29O15, 581.15010). 1H NMR (DMSO-d6, 500 MHz): d 12.68(1H, s, 5-OH), 7.34 (1H, d, J = 2.0 Hz, H-20), 7.25 (1H, dd, J = 2.0,8.0 Hz, H-60), 6.88 (1H, d, J = 8.0 Hz, H-50), 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-10 0), 4.14 (1H,d, J = 7.5 Hz, H-10 0 0), 4.04 (1H, d, J = 2.0 Hz, H-20 0), 0.89 (3H, d,J = 6.5 Hz, H-rha-CH3). 13C NMR (DMSO-d6, 125 MHz): d 177.8 (C-4), 164.3 (C-7), 161.3 (C-5), 156.9 (C-9), 156.4 (C-2), 148.7 (C-40),145.2 (C-30), 134.2 (C-3), 120.8 (C-10), 120.4 (C-60), 115.5 (C-50),115.4 (C-20), 106.4 (C-10 0 0), 103.9 (C-10), 100.9 (C-10 0), 98.7 (C-6),93.6 (C-8), 80.6 (C-20 0), 76.2 (C-30 0 0), 73.7 (C-20 0 0), 71.7 (C-40 0), 70.3(C-30 0), 70.2 (C-40 0 0), 69.3 (C-50 0), 65.7 (C-50 0 0), 17.4 (C-60 0).

N-trans-feruloyltyramine (peak 26): amorphous white powder,ESI-HRMS m/z: 314.13864 [M+H]+ (calcd for C18H21O4N,314.13868). 1H NMR (DMSO-d6, 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-40, H-80), 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-50, H-70), 6.43 (1H, d,J = 16.2 Hz, H-2), 3.80 (3H, s, 6-OCH3), 3.32 (2H, m, H-10), 2.64(2H, t, J = 7.2 Hz, H-20). 13C NMR (DMSO-d6, 150 MHz): d 165.2(C-1), 155.6 (C-60), 148.2 (C-7), 147.8 (C-6), 138.8 (C-3), 129.4 (C-30, 40, 80), 126.4 (C-4), 121.5 (C-9), 119.0 (C-2), 115.6 (C-8), 115.1(C-50, 70), 110.7 (C-5), 55.5 (-OCH3), 40.6 (C-10), 34.4 (C-20).

3.3.2. 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 wave-length, accurate molecular weight and multiple-stage mass datawere obtained in Table 3 and their fragmentation pathways wereproposed.

3.3.2.1. Quinic acid derivatives. Chlorogenic acid (Peak 6, tR = 12.82 -min) displayed maximum UV absorption at 234 nm and 325 nm,[M+H]+ ion at m/z 355. The MS2 spectrum gave the base peak atm/z 163 [caffeic acid – H2O + H]+, due to the loss of a quinic acidmoiety (192 Da). In the MS3 mass spectrum, ion at m/z 163 furtherfragmented to ion at m/z 145 [caffeic acid – 2H2O + H]+, which wasattributed to the loss of 18 Da. Then, fragment ion at m/z 117 [caf-feic acid – 2H2O – CO + H]+ was observed in the MS4 mass spec-trum, which resulted from the ion at m/z 145 for the loss of 28 Da.

Table 3Identification of individual peaks from tartary buckwheat by FTICR-MS and ESI-MSn.

Peak no. tR

(min)Observedmass (Da)

[M+H]+ or[M+Na]+

Calculatedmass (Da)

Error(ppm)

k max(nm)

ESI-MSn data (relative intensity, %) Identification and relativecontent (%)

1 6.63 205.09721 C11H13O2N2 205.09715 0.27 229,262 MS2 [205]: 188(100) Tryptophaneb(0.02%)MS3 [205 ? 188]: 146(100), 144(12)MS4 [205 ? 188 ? 146]: 118(100)

2 9.81 453.13916 C21H25O11 453.13914 0.05 232,278 MS2 [453]: 435(11), 417(11), 301(68),291(100),

(epi) Catechin-hexoseb

283(10), 273(17) (0.04%)MS3 [453 ? 291]: 273(24), 165(44),151(23),139(100), 123(96)

3 10.59 579.14984 C30H27O12 579.14970 0.24 – MS2 [579]: 427(100), 411(11), 409(67),301(18),

Procyanidin B3b

291(46), 289(24), 247(23) (<0.01%)MS3 [579 ? 427]: 409(100), 301(68),287(18),275(66), 247(12), 139(12)MS3 [579 ? 409]: 299(16), 287(100),283(26),271(16), 259(34)

4 11.31 579.14978 C30H27O12 579.14970 0.13 233,278 MS2 [579]: 453(23), 439(10), 435(10),427(100),

Procyanidin B1b

411(10), 409(55), 301(19), 289(14),247(16)

(0.02%)

MS3 [579 ? 427]: 409(100), 301(84),287(12),275(35), 247(11), 139(11)MS3 [579 ? 409]: 391(13), 299(17),287(100),283(13), 259(60), 257(17), 245(10)

5 11.93 291.08633 C15H15O6 291.08331 0.05 236,279 MS2 [291]: 273(22), 165(49), 151(26),123(100)

Catechina(0.7%)

139(97),MS3 [291 ? 123]: 95(100)MS3 [291 ? 139]: 111(100), 93(18),67(19)

6 12.82 355.10226 C16H19O9 355.10236 �0.28 234,325 MS2 [355]: 163(100) Chlorogenic acida

MS3 [355 ? 163]: 145(100), 135(10) (4.75%)MS4 [355 ? 163 ? 145]: 117(100)

7 14.72 579.14966 C30H27O12 579.14970 �0.07 237,278 MS2 [579]: 427(89), 409(25), 301(10),291(100),

Procyanidin B4b(0.46%)

289(89), 271(10)8 15.89 579.14978 C30H27O12 579.14970 0.13 235,278 MS2 [579]: 427(100), 411(14), 409(50),

301(19),Procyanidin B2b(0.03%)

291(50), 289(13), 247(20)MS3 [579 ? 427]: 409(100), 301(79),287(13)275(50), 247(13), 139(10)MS3 [579 ? 409]: 299(19), 287(100),283(29),271(13), 259(25)MS4 [579 ? 427 ? 409]: 287(100)

9 16.84 291.08633 C15H15O6 291.08331 0.05 234,279 MS2 [291]: 273(21), 165(46), 151(27),139(97),

Epicatechina(0.04%)

123(100)MS3 [291 ? 123]: 95(100)MS3 [291 ? 139]: 111(100), 93(10),67(21)

10 17.00 339.10754 C16H19O8 339.10744 0.28 233,285 MS2 [339]: 147(100) Coumaroyl quinic acidb

MS3 [339 ? 147]: 119(100) (2.3%)11 18.12 353.12320 C17H21O8 353.12309 0.30 237,277 MS2 [353]: 191(100) Methyl coumaroyl quinic

acidb(0.17%)MS3 [353 ? 191]: 191(100), 176(11),163(35)

12 19.46 369.11807 C17H21O9 369.11801 0.17 239,283 MS2 [369]: 177(100) Feruloyl quinic acidb

MS3 [369 ? 177]: 145(100) (1.05%)MS4 [369 ? 177 ? 145]: 117(100)

13 19.73 865.19775 C45H37O18 865.19744 0.36 234,277 MS2 [865]: 713(95), 695(45), 533(100),287(15)

Procyanidin trimer

MS3 [865 ? 533]: 515(25), 407(78),287(100),

A-typeb(0.20%)

247(33)MS3 [865 ? 713]: 695(100), 561(25),543(42),409(14), 287(19)MS4 [856 ? 713 ? 695]: 677(12),569(13),

(continued on next page)


Table 3 (continued)

Peak no. tR


[M+H]+ or[M+Na]+

Calculatedmass (Da)

Error(ppm)

k max(nm)


409(100), 287(25), 257(17)14 20.74 865.19751 C45H37O18 865.19744 0.08 234,277 MS2 [865]: 713(100), 695(46), 575(10),

533(98),Procyanidin trimer

411(11), 287(16) A-typeb(0.41%)MS3 [865 ? 533]: 515(18), 407(64),287(100),247(29)MS3 [865 ? 713]: 695(100), 561(23),543(44),427(11), 409(16), 287(26)MS4 [856 ? 713 ? 695]: 569(19),543(53),409(100), 287(28), 257(10)

15 28.15 443.09747 C22H19O10 443.09727 0.44 – MS2 [443]: 291(10), 273(100), 151(15),139(11)

(epi) Catechin gallateb

MS3 [433 ? 273]: 151(16), 147(15),123(100)

(<0.01%)

16 28.27 611.16077 C27H31O16 611.16066 0.18 255,355 MS2 [611]: 465(25), 303(100) Rutina(0.02%)MS3 [611 ? 303]: 303(13), 285(49),274(12),257(100), 247(35), 229(67), 165(53),153(20), 137(14)MS3 [611 ? 465]: 447(14), 303(100)MS4 [465 ? 303]: 303(11), 285(64),274(11),257(100), 247(34), 229(25),165(53),137(11)

17 28.71 465.10287 C21H21O12 465.10275 0.25 255,354 MS2 [465]: 303(100) Quercetin-3-O-b-D-galactosidea(1.41%)MS3 [465 ? 303]: 303(14), 285(57),

274(13),257(100), 247(32), 229(56), 165(44),153(14), 137(15)

18 29.16 479.08221 C21H19O13 479.08202 0.40 246,340 MS2 [479]: 303(100) Quercetin-O-glucuronideb

MS3 [479 ? 303]: 303(14), 285(60),275(14),

(2.01%)

274(15), 257(100), 247(24), 229(76),165(51), 153(13), 137(20)MS4 [479 ? 303 ? 257]: 229(100)

19 29.66 465.10251 C21H21O12 465.10275 �0.52 255,353 MS2 [465]: 303(100) Quercetin-3-O-b-D-glucosidea(5.99%)MS3 [465 ? 303]: 303(10), 285(54),

275(11),274(12), 257(100), 247(28), 229(64),195(7),165(53), 153(12), 137(15)MS4 [465 ? 303 ? 257]: 229(100)

20 30.77 581.15015 C26H29O15 581.15010 0.09 265,348 MS2 [581]: 449(23), 287(100) Kaempferol-O-pentosylhexosideb(1.21%)MS3 [581 ? 287]: 287(28), 269(27),

259(12),258(23), 241(100), 231(28), 213(55),165(77), 153(30), 121(22), 111(10)

21 33.45 449.10751 C21H21O11 449.10784 �0.73 265,348 MS2 [449]: 287(100) Kaempferol-3-O-b-D-galactosidea(1.92%)MS3 [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)MS4 [449 ? 287 ? 241]: 213(100)

22 35.96 449.10764 C21H21O11 449.10784 �0.44 265,347 MS2 [449]: 287(100) Kaempferol-3-O-b-D-glucosidea(6.38%)MS3 [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)MS4 [449 ? 287 ? 241]: 213(100)

23 36.35 449.10785 C21H21O11 449.10784 0.03 255,348 MS2 [449]: 303(100) Quercetin-3-O-a-L-rhamnosidea(5.66%)MS3 [449 ? 303]: 303(17), 285(56),

274(11),257(100), 247(34), 229(69), 165(46),153(14), 137(17)MS4 [449 ? 303 ? 257]: 229(100)


Table 3 (continued)

Peak no. tR


[M+H]+ or[M+Na]+

Calculatedmass (Da)

Error(ppm)

k max(nm)


24 37.91 581.14990 C26H29O15 581.15010 �0.34 255,349 MS2 [581]: 449(100), 431(10), 303(61) Quercetin-3-O-[b-D-xyloxyl-(1 ? 2)-a-L-rhamnoside]a(7.80%)MS3 [581 ? 303]: 303(44), 285(61),

274(13),257(100), 247(33), 229(60),165(53),153(15)MS3 [581 ? 449]: 431(77), 413(100),345(29),303(11)MS4 [581 ? 449 ? 413]: 395(100),315(25)

25 38.58 419.09735 C20H19O10 419.09727 0.18 263,349 MS2 [419]: 287(100) Kaempferol-O-pentosideb

MS3 [419 ? 287]: 287(43), 269(41),241(100),

(0.51%)

231(31), 213(66), 165(90), 153(44),147(10),133(22), 121(32), 111(12)

26 45.33 314.13864 C18H21O4N 314.13868 �0.14 238,318 MS2 [314]: 177(100) N-trans-feruloyltyraminea

MS3 [314 ? 177]: 145(100) (7.71%)MS4 [314 ? 177 ? 145]: 117(100)MS5 [314 ? 177 ? 145 ? 117]:89(100)

27 46.78 611.13977 C30H27O14 611.13953 0.39 – MS2 [611]: 593(11), 345(23), 309(80),303(100), 291(28)

Quercetin-O-coumaroylhexoseb(0.84%)

MS3 [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)MS3 [611 ? 309]: 147(100), 291(90)MS4 [611 ? 309 ? 147]: 119(100)

28 47.28 611.13983 C30H27O14 611.13953 0.49 – MS2 [611]: 593(10), 345(19), 309(84),303(100),

Quercetin-O-coumaroylhexoseb(0.46%)

MS3 [611 ? 303]: 303(40), 285(100,275(30),257(81), 247(34), 229(85), 213(30),165(49), 153(28), 137(14), 111(12)MS3 [611 ? 309]: 147(100), 291(10)

29 48.78 595.14435 C30H27O13 595.14462 �0.45 – MS2 [595]: 329(18), 309(75), 287(100) Kaempferol-O-coumaroylhexoseb(1.06%)MS3 [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)MS3 [595 ? 309]: 147(100)MS4 [595 ? 309 ? 147]: 119(100)

30 49.17 595.14484 C30H27O13 595.14462 0.37 243,328 MS2 [595]: 309(69), 291(23), 287(100) Kaempferol-O-coumaroylhexoseb(2.23%)MS3 [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)MS3 [595 ? 309]: 291(93), 147(100)MS4 [595 ? 309 ? 147]: 119(100)

31 49.84 595.14496 C30H27O13 595.14462 0.58 – MS2 [595]: 309(10), 287(100), Kaempferol-O-coumaroylhexoseb(0.54%)MS3 [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)MS3 [595 ? 309]: 291(40), 147(100)MS4 [595 ? 309 ? 147]: 119(100)

32 53.13 803.21588 C39H40O17Na 803.21577 0.14 243,312 MS2 [803]: 641(100) 1, 3, 6-tri-p-coumaroylsucroseb(0.63%)MS3 [803 ? 641]: 623(100), 477(56),

459(37),405(43), 333(18), 331(23), 313(24)

33 63.28 979.26312 C49H48O20Na 979.26311 0.01 243,314 MS2 [979]: 641(100) 1, 3, 6-tri-p-coumaroyl-60-feruloyl sucrosea

MS3 [979 ? 641]: 623(100), 477(50),459(30),

(1.52%)

405(36), 333(16), 331(23), 313(31)

(continued on next page)


Fig. 3. Major mass fragmentations proposed for peak 36.

Table 3 (continued)

Peak no. tR


[M+H]+ or[M+Na]+

Calculatedmass (Da)

Error(ppm)

k max(nm)


34 63.95 1009.27393 C50H50O21Na 1009.27368 0.25 244,317 MS2 [1009]: 671(100) 3, 6-di-p-coumaroyl-1, 60-di-feruloyl sucrosea

MS3 [1009 ? 671]: 653(100), 489(24),477(25),

(2.99%)

459(6), 435(33), 363(12), 331(15),313(22)

35 64.68 1039.28284 C51H52O22Na 1039.28424 �1.35 243,322 MS2 [1039]: 701(100) 1, 6, 60-tri-feruloyl-3-p-coumaroyl sucrosea

MS3 [1039 ? 701]: 683(100), 519(20),507(32),

(4.72%)

489(11), 435(30), 363(17), 361(14),343(17)

36 65.34 1069.29431 C52H54O23Na 1069.29481 �0.47 244,327 MS2 [1069]: 731(100) 1, 3, 6, 60-tetra-feruoylsucrosea(2.47%)MS3 [1069 ? 731]: 713(100), 537(28),

519(21),465(27), 393(13), 361(10), 343(16)

–UV spectra were not available due to low intensity.a indicates the compounds were unequivocally identified.b indicates the compounds were tentatively assigned.


3.3.2.2. Flavan-3-ol derivatives. Catechin (peak 5, tR = 11.93 min)and epicatechin (peak 9, tR = 16.84 min) showed the same [M+H]+

ion at m/z 291. Both of two peaks produced the typical fragmentsof m/z 273, 165, 151, 139 and 123 from ion at m/z 291 in theMS2 spectrum, which arose from cleavage of the C ring. Then, inthe MS3 spectrum from the base peak ion at m/z 139 generatedthe characteristic fragment ions at m/z 111, 93 and 67. The frag-ment ion at m/z 123 produced the ion at m/z 95.

3.3.2.3. Flavonol derivatives. In tartary buckwheat extract, there area great number of flavonol derivatives that are active chemical con-stituents associated with antioxidant and antidiabetic activities (Liet al., 2009). The major flavonol glycosides consist of two types offlavonol aglycones such as quercetin and kaempferol. UV spectrumand multi-stage mass data of flavonol aglycone and glycoside arevaluable for characterization and identification.

Peak 16, peak 17, peak 19, peak 23 and peak 24 belong to quer-cetin aglycone.

Rutin (peak 16, tR = 28.27 min) exhibited the protonated mole-cule [M+H]+ ion at m/z 611. In the MS2 spectrum, the fragmention 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 – (146 + 162)]+ from the molecularion. The MS3 mass spectrum of m/z 303 presented the specific frag-ment ions at m/z 285, 257, 247, 229, 165 and 153, which character-ized quercetin aglycone. It was established by comparison withpreviously reported data (Verardo et al., 2010).

Quercetin-3-O-b-D-galactoside (peak 17, tR = 28.71 min) andquercetin-3-O-b-D-glucoside (peak 19, tR = 29.66 min) presentedthe same [M+H]+ ion at m/z 465. In the MS2 spectrum, the loss ofa hexose moiety (162 Da) from the ion at m/z 465 gave the frag-ment ion at m/z 303 with the highest relative intensity. Then thesimilar fragmentation pattern of quercetin aglycone from the frag-ment ion at m/z 303 was observed in the MS2 spectrum.

Quercetin-3-O-a-L-rhamnoside (peak 23, tR = 36.35 min) andquercetin-3-O-[b-D-D-xyloxyl-(1 ? 2)-a-L-rhamnoside] (peak 24,tR = 37.91 min) illustrated [M+H]+ ions at m/z 449 and at m/z 581,respectively. In the MS2 spectrum of peak 23, the fragment ion atm/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 303was similar with those of peak 16, 17 and 19 in MS3 spectrum.The [M+H]+ ion at m/z 581 of peak 24 yielded the major production at m/z 449 with the highest relative intensity and ion at m/z303 corresponding to the loss of a pentose (132 Da) moiety and


rhamnose (146 Da) moiety. The MS3 mass spectrum of peak 24 fromthe fragment ion at m/z 303 was similar to that of quercetinaglycone.

Kaempferol-3-O-b-D-galactoside (peak 21, tR = 33.45 min) andkaempferol-3-O-b-D-glucoside (peak 22, tR = 35.96 min) belongedto kaempferol aglycone and presented the same [M+H]+ ions at m/z 449. In the MS2 spectrum, the prominent fragment ion at m/z287 was assigned to the loss of a hexose moiety (162 Da) from[M+H]+ ion at m/z 449. The MS3 mass spectrum of m/z 287 illustratedthe fragment ions at m/z 269, 241, 231, 213, 165 and 153, whichwere the characteristic fragmentations of kaempferol aglycone.

In summary, two kinds of aglycones showed the differencemaximum UV absorption, quercetin glycoside at 250–260 nmand 345–355 nm and kaempferol glycoside at 260–265 nm and345–348 nm. Quercetin glycoside produced characteristic frag-ment ion at m/z 303, then further fragmented to characteristic ionsat m/z 285, 257, 247, 229, 165 and 153, whereas kaempferol glyco-side yielded diagnostic fragment ion at m/z 287, then gave rise tocharacteristic ions at m/z 269, 241, 231, 213, 165 and 153. Themost commonly encountered sugar, followed by pentose (xyloseand arabinose, 132 Da), deoxyhexose (rhamnose, 146 Da) and hex-ose (glucose and galactose, 162 Da). Two types of flavonol deriva-tives could be distinguished according to maximum UVabsorption and fragmentation pattern.

3.3.2.4. Phenylpropanoid glycosides. Phenylpropanoid glycosidesshowed the characteristic maximum UV absorption wavelengthat 220–250 nm and 310–340 nm.

1, 3, 6-tri-p-coumaroyl-60-feruloyl sucrose (peak 33, tR = 63.28 -min) presented the [M+Na]+ ion at m/z 979. The characteristic ionat m/z 641 with the highest relative intensity from [M+Na]+ ion atm/z 979 was produced in the MS2 spectrum, corresponding to thecharacteristic loss of feruloyl hexose moiety (338 Da). In the MS3

spectrum, the four major fragments at m/z 623 [641 – H2O] withthe highest relative intensity, m/z 477 [641 – H2O – p-coumaroyl],m/z 459 [641 – 2H2O – p-coumaroyl] and m/z 313 [641 – 2H2O – 2p-coumaroyl] were observed, due to the loss of H2O and p-couma-royl moiety. The characteristic fragment ions indicated that oneferuloyl moiety was located on the glucopyranose ring and threep-coumaroyl moieties were on the fructofuranose ring. Moreover,its structure was confirmed by NMR data and in agreement withthe literature (Takasaki et al., 2001).

3, 6-di-p-coumaroyl-1, 60-di-feruloyl sucrose (peak 34,tR = 63.95 min) and 1, 6, 60-tri-feruloyl-3-p-coumaroyl sucrose(peak 35, tR = 64.68 min) showed [M+Na]+ ions at m/z 1009 and1039, respectively. Peak 35 had one more methoxyl group thanpeak 34. The characteristic ion at m/z 671 and 701 with the highestrelative intensity from [M+Na]+ ion at m/z 1009 and 1039 wereproduced in the MS2 spectrum, corresponding to the characteristicloss of feruloyl hexose moiety (338 Da). The fragmentation patternof peak 34 and peak 35 were similar to peak 33.

1, 3, 6, 60-tetra-feruloyl sucrose (peak 36, tR = 65.34 min) pre-sented [M+Na]+ ion at m/z 1069. The MS2 fragmentations yieldedion at m/z 731 the highest relative intensity, corresponded to theloss of feruloyl hexose moiety (338 Da). In the MS3 spectrum, themajor fragments were m/z 713 [731 – H2O], m/z 537 [731 – H2O– feruloyl], m/z 519 [731 – 2H2O – feruloyl] and m/z 343 [731 –2H2O – 2 feruloyl], due to the loss of H2O and feruloyl moiety.The fragmentation scheme is given in Fig. 3.

The typical fragmentation pathways for phenylpropanoid glyco-sides were the loss of feruloyl hexose moiety (338 Da), H2O, p-cou-maroyl and feruloyl moiety subsequently.

3.3.2.5. Nitrogen compound. N-trans-feruloyltyramine (peak 26,tR = 45.33 min) displayed the [M+H]+ ion at m/z 314. The MS2 spec-trum showed the base peak at m/z 177, which was corresponded to

feruloyl moiety. The MS3 spectrum of ion m/z 177 produced thebase peak at m/z 145, corresponding to the loss of (CH3OH, 32 Da).

3.3.3. Tentative identification of tartary buckwheat by HPLC-PDA/LTQ-FTICRMS

By comparing the information with the isolated compounds, theothers were tentatively identified. Their structural informationwere collected and listed in Table 3.

3.3.3.1. Quinic acid derivatives. Peak 10, peak 11 and peak 12 showedsimilar fragmentation pattern to peak 6. Peak 10 (tR = 17.00 min)exhibited [M+H]+ ion at m/z 339.10754, which corresponded to themolecular formula of C16H19O8. In the MS2 spectrum, ion at m/z339 produced a base peak at m/z 147 [p-coumaric acid – H2O + H]+,corresponding to the typical loss of quinic acid moiety (192 Da).The MS3 spectrum ion at m/z 147 produced ion at m/z 119[p-coumaric acid – H2O – CO + H]+, which was attributed to the lossof 28 Da. In contrast to literatures data (Alonso-Salces et al.,2004; Jaiswal & Kuhnert, 2011; Vallverdú-Queralt et al., 2011), peak10 was tentatively identified as coumaroyl quinic acid.

Peak 11 (tR = 18.12 min) displayed [M+H]+ ion at m/z 353.12320,indicating the molecular formula of C17H21O8. In the MS/MS spec-trum, then it produced a base peak at m/z 191, corresponding tothe loss of 162 Da [caffeic acid – H2O]. The MS3 spectrum of ionat m/z 191 yielded ions at m/z 176 and 163. Peak 11 was tentativelyidentified as methyl coumaroyl quinic acid (Rakesh & Nikolai,2011).

Peak 12 (tR = 19.46 min) exhibited [M+H]+ ion at m/z 369.11807,corresponding to the molecular formula of C17H21O9. In the MS/MSspectrum gave the major ion at m/z 177 [ferulic acid – H2O + H]+,corresponding to the loss of quinic acid moiety (192 Da). TheMS3 spectrum of m/z 177 produced ion at m/z 145 [ferulicacid – H2O – CH3OH + H]+. The multi-stage mass data were in goodagreement with the literatures (Jaiswal & Kuhnert, 2011;Vallverdú-Queralt et al., 2011). Peak 12 was tentatively identifiedas feruloyl quinic acid.

3.3.3.2. Flavan-3-ol derivatives. Peak 2 (tR = 9.81 min) showed max-imum UV absorption wavelength at 232 nm and 278 nm, [M+H]+

ion at m/z 453.13916, corresponding to C21H25O11. The MS2 spec-trum of ion at m/z 453 gave a base peak at m/z 291, correspondingto the loss of a hexose moiety (162 Da). The MS3 mass spectrum offragment ion at m/z 291 produced the characteristic fragments ofm/z 273, 165, 151, 139 and 123, which were similar to those of(epi) catechin. Above all these data matched well with the litera-ture (Verardo et al., 2010). Thus, peak 2 was tentatively identifiedas (epi) catechin-hexose.

In nature the dimeric procyanidins exist as the B-type procyani-dins, which consist of four major isomers such as B1, B2, B3 and B4

(Pekic, Kovac, Alonso, & Revilla, 1998; Sun & Miller, 2003). Theseisomers indicate the similar UV absorbance and same elementalcomposition. Multi-stage data demonstrates the similar fragmen-tation ions and relative intensities. However, the difference onretention time is likely to identify the procyanidins due to linkageor stereochemistry. Generally, the procyanidin B2 on C18 column isat the retention time between catechin and epicatechin, near toepicatechin (Verardo et al., 2010). In addition, the B-type procyani-dins such as B3, B1, B4 and B2 were eluted sequence on C18 column(Pekic et al., 1998; Sun & Miller, 2003).

Peak 3 (tR = 10.59 min), peak 4 (tR = 11.31 min) and peak 7(tR = 14.72 min) exhibited [M+H]+ ions at m/z 579.14984 (calcdfor C30H27O12), 579.14978 (calcd for C30H27O12) and 579.14966(calcd for C30H27O12), respectively. The three peaks mass spectrumpresented the specific fragments of m/z 427 and 409, which werethe characteristic fragmentations in agreement with procyanidinB-type dimmers, by comparison with previously reported data


(Pekic et al., 1998; Sarnoski, Johnson, Reed, Tanko, & O’Keefe, 2012;Soong & Barlow, 2005; Sun & Miller, 2003; Verardo et al., 2010;Wollgast, Pallaroni, Agazzi, & Anklam, 2001). The ion at m/z 427[M – 152]+ originated from retro Diels–Alder fragmentation ofthe heterocyclic ring. In the MS3 mass spectrum of peak 3 and peak4 from ion at m/z 427 produced the fragments at m/z 409, 301, 287,275, 247 and 139. The ion at m/z 409 yielded the fragments at m/z299, 287, 283 and 259. Peak 7 could not obtain the multiple-stagemass data for low concentration. Thus peak 3, peak 4 and peak 7were tentatively identified as procyanidin B-type dimmers suchas B3, B1 and B4.

Peak 8 (tR = 15.89 min) exhibited [M+H]+ ion at m/z 579.14978(calcd for C30H27O12). The MS2 spectrum presented the typical frag-ment ions at m/z 427, 409 and 291 from ion at m/z 579. The cleav-age of inter-flavanoid C–C linkage obtained the fragment at m/z291, corresponding to the loss of (epi) catechin unit 288 Da fromion at m/z 579. The MS3 spectrum of m/z 427 [M – 152]+ producedthe fragments of m/z 409, 301, 287, 275, 247 and 139. The MS3

mass spectrum of ion at m/z 409 produced the base peak ion atm/z 287 from ion at m/z 427. On the base of above the data, peak8 was tentatively identified as procyanidin B2 (Alonso-Salceset al., 2004).

Peak 13 (tR = 19.73 min) and peak 14 (tR = 20.74 min) showedmaximum UV absorption wavelength at 234 nm and 277 nm,[M+H]+ ions at m/z 865.19775 (calcd for C45H37O18) and865.19751 (calcd for C45H37O18), respectively. Both of them pro-duced major fragments at m/z 713, 695 and 533 in the MS2 spec-trum. The ion at m/z 713 originated from retro Diels–Alderfragmentation of the heterocyclic ring, corresponding to the lossof galloyl moiety (152 Da). In the MS3 spectrum, the ion at m/z533 produced the fragments at m/z 515, 407, 287 and 247 fromm/z 865; the fragment ion m/z 713 yielded the fragments at m/z695, 561, 543, 409 and 287. The MS4 mass spectrum of ion atm/z 695 from ion at m/z 713 gave the fragments at m/z 569, 409,287 and 257. These fragments were in accordance with the litera-tures (He, Pan, Shi, Zhang, & Duan, 2009; Sarnoski, Johnson, Reed,Tanko, & O’Keefe, 2012; Wollgast et al., 2001). Therefore, peak 13and peak 14 were tentatively identified as procyanidin trimerA-type.

Peak 15 (tR = 28.15 min) exhibited [M+H]+ ion at m/z 443.09747,which corresponded to the molecular formula of C22H19O10. TheMS2 mass spectrum of ion at m/z 443 produced the fragments atm/z 291, 273, 151 and 139. The fragment ion at m/z 291 was dueto the loss of galloyl moiety (152 Da) from ion at m/z 443.The otherfragment ions at m/z 273, 151 and 139 was similar to the MS2 massspectrum of peak 5 and peak 9. Therefore, this peak was tentativelyidentified as (epi) catechin-gallate (Verardo et al., 2010).

3.3.3.3. Flavonol derivatives. Peak 18 (tR = 29.16 min) with a [M+H]+

ion at m/z 479.08221(calcd for C21H19O13), it generated the frag-ment ion at m/z 303 corresponding to loss of the glucuronide moi-ety (176 Da) in the MS2 spectrum. The fragment ions at m/z 285,257, 247, 229, 165 and 153 in the MS3 spectrum were observedfrom ion at m/z 303, which were assigned to quercetin aglycone.Therefore, peak 18 was tentatively identified as quercetin glucuro-nide (Irene, Gian, & Antonio, 2004).

Peak 20 (tR = 30.77 min) presented maximum UV absorptionbands at 265 nm and 348 nm, [M+H]+ ion at m/z 581.15015 (calcdfor C26H29O15). In the MS/MS spectrum, the two major fragmentswere ions at m/z 449 and 287 from ion at m/z 581, which were cor-responded to the loss of a pentose moiety [M – 132]+ and a hexosemoiety [M – (132 + 162)]+, respectively. The MS3 mass spectrum ofion at m/z 287 presented the characteristic fragments at m/z 287,269, 241, 231, 213, 165 and 153 for kaempferol aglycone. For thelack of both reference substance and reported data, assignmentto the position of pentose and hexose moieties was not possible.

Thus, this peak was tentatively identified as kaempferol-O-pento-syl hexoside.

Peak 25 (tR = 35.58 min), it was detected at [M+H]+ m/z419.09735 (calcd for C20H19O10). In the MS2 spectrum, the majorfragment ion m/z 287 was corresponded to the loss of a pentosemoiety [M – 132]+ from protonated molecule. The MS3 mass spec-trum of m/z 287 presented the specific fragments of m/z 287, 269,241, 231, 213, 165 and 153, which was the diagnostic fragmenta-tion for kaempferol aglycone. Therefore, this peak was tentativelyidentified as kaempferol-O-pentoside.

The two isomers of peak 27 (tR = 46.78 min) and peak 28(tR = 47.28 min) had [M+H]+ at m/z 611.13977 (calcd forC30H27O14) and 611.13983 (calcd for C30H27O14), respectively. Thetwo peaks had stronger retention on the C18 column than querce-tin-O-dihexoside. The MS2 fragmentations from [M+H]+ yieldeddiagnostic ion at m/z 303 assigned to quercetin aglycone fragmen-tations, and ion at m/z 309 corresponding to [coumaryol hex-ose + H]+. In the MS3 spectrum, the ion at m/z 147 was the basepeak from the ion at m/z 309, which was corresponded to coumaricacid. In the MS3 spectrum of these two isomers, ion at m/z 291 fromion at m/z 309 showed the difference relative intensity. The relativeintensity of ion at m/z 291 in the peak 27 was higher than that ofthe peak 28. Hence, according to the reported data (Ye, Yan, &Guo, 2005), the two isomers of peak 27 and 28 were tentativelyidentified as quercetin-O-coumaroyl hexose.

The three isomers of peak 29 (tR = 48.78 min), peak 30(tR = 49.17 min) and peak 31 (tR = 49.84 min) had the [M+H]+ atm/z 595.14435 (calcd for C30H27O13), 595.14484 (calcd forC30H27O13) and 595.14496 (calcd for C30H27O13). UV absorbancewavelength of peak 29 and peak 31 could not be detected becauseof low concentration. The MS2 fragmentations of peak 30 yieldedthe two major ions at m/z 309 corresponding to the [coumaryolhexose + H]+ and ion at m/z 287 assigned to kaempferol aglyconefragmentations. In the MS3 spectrum, the ion m/z 147 was the basepeak from ion at m/z 309, corresponding to coumaric acid. The ionat m/z 595 of the three isomers generated the two same ions at m/z309 and 287 in the MS2 spectrum. The ion at m/z 309 of peak 30and peak 31 gave the ion at m/z 291 with the difference relativeintensities in the MS3 spectrum, which was consistent with the lit-erature (Ye et al., 2005). Peak 29, peak 30 and peak 31 were tenta-tively identified as kaempferol-O-coumaroyl hexose.

3.3.3.4. Phenylpropanoid glycosides. Peak 32 (tR = 53.13 min) pre-sented maximum UV absorption wavelength at 243 nm and312 nm, [M+Na]+ ion at m/z 803.21588 (calcd for C39H40O17Na).The MS2 fragmentation yielded the major ion at m/z 641 fromion at m/z 803, which was corresponded to the loss of hexose moi-ety (162 Da). The characteristic fragment ion indicated that non-substitution was located on the glucopyranose ring. In the MS3

spectrum, ion at m/z 641 produced the four major fragment ionsat m/z 623 [641 – H2O], 477 [641 – H2O – p-coumaroyl], 459[641–2H2O – p-coumaroyl] and 313 [641–2H2O – 2 p-coumaroyl],corresponding to the loss of H2O and p-coumaroyl moiety. There-fore peak 32 was tentatively identified as 1, 3, 6-tri-p-coumaroylsucrose on the base of the fragmentation pattern of phenylpropa-noid glycosides.

3.3.3.5. Nitrogen compounds. Peak 1 (tR = 6.63 min) displayed max-imum UV absorption wavelength at 229 nm and 262 nm, [M+H]+

ion at m/z 205.09721 (calcd for C11H13O2N2). The MS2 spectrumof ion at m/z 205 yielded ion at m/z 188, corresponding to the lossof amino moiety (17 Da). The MS3 spectrum of ion at m/z 188 pre-sented the base peak at m/z 146, corresponding to the loss ofCO + CH2 (42 Da). The MS4 spectrum of ion at m/z 146 yieldedthe major ion at m/z 118, which was corresponded to the loss of


CO moiety (28 Da). Therefore, peak 1 was tentatively identified astryptophane.

3.4. Relative contents of flavonoids and phenylpropanoid glycosides

Chromatographic peak area normalization method was used forcalculating their relative content of compositions. Vertical splittingoverlap peaks were used to modify the peaks integration. The rel-ative contents of identified compounds had been listed in Table 3.The flavonoids and phenylpropanoid glycosides were main constit-uents as shown in Fig. 2, and the relative contents of total identi-fied flavonoids were 38.04%. Meanwhile, the most predominantflavonoids were quercetin-3-O-b-D-glucoside (peak 19), kaempfer-ol-3-O-b-D-glucoside (peak 22), quercetin-3-O-a-L-rhamnoside(peak 23), and quercetin-3-O-[b-D-xyloxyl-(1 ? 2)-a-L-rhamno-side] (peak 24) representing approximately 25.83%. The relativecontents of other major phenlypropanoid glycosides were approx-imately 11.7%. The ratio of four main phenlypropanoid glycosidesincluding 1, 3, 6-tri-p-coumaroyl-60-feruloyl sucrose (peak 33), 3,6-di-p-coumaroyl-1, 60-di-feruloyl sucrose (peak 34), 1, 6, 60-tri-feruloyl-3-p-coumaroyl sucrose (peak 35), 1, 3, 6, 60-tetra-feruloylsucrose (peak 36) was nearly 1:2:3:2.

4. Conclusion

The result demonstrated that HPLC-PDA/LTQ-FTICRMS methodwas established to profile the chemical constituents of tartarybuckwheat. A total of 36 compounds were identified on the basisof characteristic UV absorbance wavelength, diagnostic mass dataand fragmentation pathway. 11 major chemical constituents wereisolated and confirmed by spectroscopic methods. In addition, 1, 3,6, 60-tetra-feruloyl sucrose was a new phenlypropanoid glycoside.3, 6-di-p-coumaroyl-1, 60-di-feruloyl sucrose, 1, 6, 60-tri-feruloyl-3-p-coumaroyl sucrose, N-trans-feruloyltyramine and quercetin-3-O-[b-D-xyloxyl-(1 ? 2)-a-L-rhamnoside] were isolated and identifiedfrom the Fagopyrum species for the first time. At the same time, thecharacteristic fragmentation behaviors of phenylpropanoid glyco-sides were proposed and provided detailed data for on-line struc-tural identification of unknown compounds from tartarybuckwheat. HPLC-PDA/LTQ-FTICR MS method was firstly intro-duced to be applied for the phytochemical analysis of tartary buck-wheat. It is very powerful to rapidly and comprehensively probethe chemical compositions from tartary buckwheat in a singlerun. The rich chemical information is base for good utilizationand quality control of tartary buckwheat.

Acknowledgment

We gratefully acknowledge Professor Wenyi He and Yan Wu forNMR measurements.

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