Identification of phenolic compounds in Equisetum giganteum by LC–ESI-MS/MS and a new approach to total flavonoid quantification

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  Identification of phenolic compounds in Equisetum giganteum by LC–ESI-MS/MS and a new approach to total flavonoid quantification
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  Identification of phenolic compounds in  Equisetum giganteum by LC–ESI-MS/MS and a new approach to total flavonoid quantification Leandro N. Francescato a, n , Silvia L. Debenedetti b , Thiago G. Schwanz c , Valquiria L. Bassani a ,Ame´lia T. Henriques a a Programa de Po´s-Graduac   - ~ ao em Ciˆencias Farmac ˆeuticas, Faculdade de Farma´cia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil b Facultad de Ciencias Exactas y Naturales, Universidad de Belgrano, C1426DQG, Buenos Aires, Argentina c Nu´cleo de Ana´lises e Pesquisas Org ˆanicas–NAPO, Departamento de Quı´mica, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil a r t i c l e i n f o  Article history: Received 28 August 2012Received in revised form26 November 2012Accepted 26 November 2012Available online 2 December 2012 Keywords:Equisetum giganteum LC-MS/MSFlavonoidsStyrylpyronesAglyconesTotal flavonoid determination a b s t r a c t Equisetum giganteum  L., commonly called ‘‘giant horsetail’’, is an endemic species of Latin America.Its aerial parts have been widely used in ethnomedicine as a diuretic and in herbal medicine and foodsupplements as a raw material. The phenolic composition of   E. giganteum  stems was studied by liquidchromatography coupled to diode array detection (LC–DAD) and liquid chromatography coupled toelectrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS), which identified caffeic acid derivatives,flavonoids and styrylpyrones. The most abundant glycosilated flavonoids in this sample were kaempferolderivatives. Other rare phenolic components, namely, quercetin-3- O -(caffeoyl)-glucoside and 3-hydroxy-hispidin-3,4 0 -di- O -glucoside, were reported for first time in the  Equisetum  genus. An LC-UV method for thesimultaneous quantification of flavonoid aglycones in  E. giganteum  obtained after hydrolysis was developedand validated. The method exhibited excellent linearity for all analytes, with regression coefficients above0.998, LOD Z 0.043 m g mL   1 , LOQ  Z 0.158 m gmL   1 and recovery rates of 96.89–103.33% and 98.22–102.49%for quercetin and kaempferol, respectively. The relative standard deviation for the intra- and inter-dayprecision was r 3.75%. The hydrolysis process was optimized by central composite rotational design andresponse surface analysis. The second-order response models for the aglycones contents were as follows:quercetin ( m g g  1 ) ¼ 24.8102 þ 55.2823  HCl þ 0.776997  Time  7.23852  HCl 2  7.46528E  04  Time 2   0.229167  HCl  Time; kaempferol ( m g g  1 ) ¼ 9.66755 þ 974.822  HCl þ 11.8059  Time  130.612  HCl 2  0.0125694  Time 2  3.22917  HCl  Time, with estimated optimal conditions of 1.18M HCl and205min of hydrolysis. The results obtained with these new methods were compared to those from aspectrophotometric assay used to determine the total flavonoids in the  Equisetum arvense  monograph(Horsetail, British Pharmacopoeia 2011). For all four species analyzed ( E. giganteum ,  E. arvense ,  E. hyemale  and E. bogotense ), the calculated aglycone content was higher using the optimized hydrolysis conditions.Additionally, the LC method was more appropriate and specific for quantitative analysis. &  2012 Elsevier B.V. All rights reserved. 1. Introduction Equisetum giganteum  L. (Equisetaceae, subgenus  Hippochaete ),commonly known as ‘‘cavalinha’’, ‘‘cola de caballo’’, ‘‘horsetail’’ or‘‘giant horsetail’’, is a lower vascular plant widespread in Southernand Central America. This species is used in traditional medicine inMexico, Guatemala, Venezuela, Argentina and other countries, mainlyfor its diuretic, astringent, hemostatic and remineralizing properties.It is also used to treat liver and urinary disorders, among otherapplications [1–5]. In southern Brazil and Argentina,  E. giganteum infusions are often used as a diuretic and for weight loss. The in vivodiuretic activity of the extracts of this species has been verified [3,4], and no oral acute toxicity was observed in mice [1].In Brazil and Argentina, the drug is widely used and commercia-lized as a raw material for herbal medicines and as a food supple-ment. In these and other Latin American countries,  E. giganteum  iscommonly used as a substitute for  E. arvense  (Horsetail herb, Equisetiherba),aEuropeanspecieswithconfirmeddiureticactivityanda longhistory of clinical use as well as a well-known phytochemical profile[6,7]. Moreover, several pharmacopoeias include monographs of  E. arvense . In the case of   E. giganteum , the only known data aboutits chemical composition have been obtained from metal and silicacontent analyses, ash determination [5] and oleoresin analysisemploying gas chromatography–mass spectrometry [8]. No data onits phytochemical composition have been published.Considering the wide use of these plants as a raw material inherbal medicine, it is necessary to develop reliable quality control Contents lists available at SciVerse ScienceDirectjournal homepage: www.elsevier.com/locate/talanta Talanta 0039-9140/$-see front matter  &  2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.talanta.2012.11.072 n Corresponding author. Tel.:  þ 55 51 3308 5258; fax:  þ 55 51 3011 5050. E-mail address:  leandrofrancescato@yahoo.com.br (L.N. Francescato).Talanta 105 (2013) 192–203  methods to comply with regulatory requirements. For this pur-pose, the pharmacognostic parameters for  E. giganteum  have beenexamined [9]. Thus, the aim of this study was to determine, forthe first time, the phenolic profile of   E. giganteum  using liquidchromatography coupled with tandem mass spectrometry (LC–MS/MS) and to develop and validate a liquid chromatographycoupled with ultraviolet detection (LC–UV) method for thequantitation of flavonoid aglycones, including a previous statis-tical optimization of the acid hydrolysis of their glycosides. Four Equisetum  species were evaluated:  E. giganteum ,  E. arvense , E. hyemale  and  E. bogotense . The results obtained using thismethod were compared to those obtained using the hydrolysisand spectrophotometric method described for  E. arvense  in theBritish Pharmacopoeia 2011 [10]. 2. Materials and methods  2.1. Plant material The aerial sterile stems of   Equisetum giganteum  L. werecollected in Santo Antˆonio da Patrulha (RS, Brazil) in May 2011(sample 1) and October 2009 (sample 2) [9].  E. hyemale  L. wascollected in August 2009 in Curitiba (PR, Brazil).  E. bogotense H.B.K. was collected in August 2008 in Bariloche (Rı´o Negro,Argentina). A commercial sample of   E. arvense  L. was purchased inSpain, having been bottled in July 2008. All samples wereidentified botanically. The material was dried and ground to aparticle size  o 0.355 mm. Loss on drying was determined at105  1 C for 4 h.  2.2. Chemicals and reagents Distilled water and analytical-grade reagents were employedin the spectrophotometric and hydrolysis experiments: 37%hydrochloric acid from Quimex (SP, Brazil) and methanol, ethylacetate and anhydrous sodium sulfate from Synth (SP, Brazil).Ethanol (95%, Synth, SP, Brazil), ultra-pure water (Millipore, MA,USA) and HPLC-grade acetonitrile, methanol and formic acid 96%(Tedia, OH, USA) were used for chromatographic analysis.Analytical-grade standards of quercetin (Janssen Chimica,Belgium), kaempferol (Chromadex, CA, USA) and astragalin (97%,Sigma-Aldrich, MO, USA) were used as reference compounds.  2.3. LC–MS/MS and LC–DAD qualitative analysis 2.3.1. Sample preparation The powdered material of   E. giganteum  (0.4 g, sample 1) wasextracted with 2 mL of 50% aqueous ethanol (v/v) for 30 min bysonication, centrifuged (1500  g  ) and filtered (0.22 m m PVDF, Milli-pore, MA, USA). The obtained extract was diluted with ultrapurewater (1:5) prior to use in LC–DAD and LC–MS analysis.  2.3.2. Instruments LC-MS/MS analysis wasperformed on an Agilent 1200 series HPLC(Palo Alto, CA, USA) equipped with a G1312B SL binary pump, aG1367D high-performance auto-sampler (HiP ALS SL  þ ) and aG1316B SL thermostated column compartment. The mass spectro-meter was an Agilent model G6460A triple quadrupole instrumentequipped with an electrospray ionization source. Instrument control,data acquisition and processing were performed using MassHunterworkstation software (Qualitative Analysis, version B.03.01, Agilent).A Phenomenex Luna C18(2) column (250  4.6mm 2 i.d., 5 m mparticle; Torrance, CA, USA) was used.A Waters Alliance 2690 Chromatograph Separations Moduleequipped with a multiple-UV-wavelength photo-diode arraydetector (Model 996, Waters, Milford, MA, USA) was used forliquid chromatography coupled to diode array detection(LC–DAD) analysis. UV spectra were recorded in a range of 210–400 nm and monitored at 254 nm. Instrument controland data acquisition were performed using Waters EmpowerSoftware 2002.  2.3.3. LC–MS/MS and LC–DAD conditions and parameters Samples were eluted with a gradient of previously degassed0.3% (v/v) formic acid in water (eluent A, pH 2.2) and acetonitrile(eluent B). The gradient profile was 9–15% B (0–21 min), 15–22%B (21–45 min), 22–35% B (45–60 min), 35–90% B (60–65 min) and90% B (65–70 min). Separation was carried out at a flow rate of 0.9 mL min  1 at 30  1 C and the injection volume was 10 m L.Mass spectra of the column eluate were recorded in the range m/z   100–1000. The instrument was operated with a capillaryvoltage of 3500 V and a nozzle voltage of 500 V. Nitrogen wasused as the nebulizer gas at 45 psi, a carrier gas of 6 L min  1 at350  1 C and a sheath gas of 11 L min  1 at 350  1 C. MS data wereacquired in negative and positive ionization modes for theaccurate determination of the  m/z   of the parent ions. MS/MS datawere acquired in negative ionization mode to obtain the  m/z   of the fragment ion. For structural interpretation, the MS/MSfragment ions were acquired using different collision-induceddecomposition energies (CID) of 10, 20, 30, 50 and 70 V with afragmentor voltage of 135 V. The classical nomenclature proposedby Domon and Costello [11] in the MS/MS spectra of glycosideswas adopted to name the fragment ions.For LC–DAD analysis, the same chromatographic parametersdescribed for LC-MS/MS analysis were employed, with the excep-tion of an injection volume of 15 m L.  2.4. LC–UV quantitative analysis 2.4.1. Preparation of standard and sample solutions for LC–UV method validation Quercetin and kaempferol standards were dissolved in metha-nol and diluted to give seven concentrations in the range of 0.175–43.7 m g mL   1 and 0.222–55.6 m g mL   1 , respectively. E. giganteum  (sample 2) was hydrolyzed in 1.8 M HCl for120 min, extracted and dissolved in methanol according theconditions described below (2.4.5).  2.4.2. Instruments For the quantitative determination, LC–UV analysis, a WatersAlliance 2695 Chromatograph Separations Module equipped witha UV/VIS detector (Model 2487, Waters) was used. The LunaC18(2) column (4.6  250 mm 2 , 5 m m, Phenomenex, CA, USA)was protected by a Bondapak C18 guard-column (1  8 mm 2 ,37–55 m m, Waters, MA, USA). To evaluate the specificity, amultiple-UV-wavelength photo-diode array detector (Model996, Waters) was also used. Empower Software 2002 (Waters)was used for instrument control, data acquisition and processingof the chromatographic information.  2.4.3. LC–UV conditions and parameters The separation of aglycones was achieved using a lineargradient of water (A) and methanol (B), both acidified with 0.3%formic acid (v/v). The gradient profile was 47–60% B (0–15 min),60% B (15–23 min), 60–100% B (23–24 min) and 100% B (24–27 min). At the end of each analysis, the column was stabilized for10 min under the initial conditions. A volume of 10 m L of thesample was eluted with a flow rate of 0.9 mL min  1 and detectedat 370 nm; the column temperature was 22 7 2  1 C. L.N. Francescato et al. / Talanta 105 (2013) 192–203  193   2.4.4. LC–UV method validation The method linearity, precision (repeatability and intermedi-ary precision), accuracy (recovery), specificity, detection andquantitation limits were evaluated according to the ICH guide-lines [12]. Linearity : Linearity was determined by the calibration curvesobtained from the LC analysis of the standard solutions of quercetin and kaempferol. The linearity analysis, over a 3 dayperiod,was estimated byregressionusingthe leastsquaresmethod.Seven concentrations of quercetin and kaempferol in the range of 0.175–43.7 m g mL   1 and 0.222–55.6 m g mL   1 , respectively, wereemployed.  Accuracy : The accuracy was determined by recovery analysis.Measured amounts of quercetin (0.091, 0.272 and 0.453 m g mL   1 )and kaempferol (1.08, 3.24 and 5.4 m g mL   1 ) were added to thehydrolyzed extract solution to create solutions with 80, 100 and120% of the theoretical concentration. Each sample was injectedthree times and the amount recovered was calculated. Controlsfrom all samples were prepared and analyzed. Precision : To evaluate the repeatability and intermediate pre-cision (intra- and inter-day precision), five concentration levels(50 to 150%) of the hydrolyzed extract were prepared and injectedin triplicate on three different days. A new sample solution wasprepared each day. The results were expressed as relative stan-dard deviation (RSD, %). Specificity : The specificity was determined by peak purity testsusing a diode array detector after adding a small amount of thestandard substances to the sample. Limit of detection and limit of quantitation : The limit of detec-tion (LOD) and limit of quantitation (LOQ) were defined as signal-to-noise ratios of 3.3:1 and 10:1, respectively. The standardsolutions of quercetin and kaempferol for LOD and LOQ wereprepared by sequential dilution. Robustness : A Plackett–Burman (P–B) design was employed totest the robustness of the method. Four factors were tested:column (two batch and age), formic acid concentration (0.28,0.3 and 0.32%, v/v), percentage of MeOH in the initial mobilephase composition (46, 47 and 48%) and flow rate (0.85, 0.9 and0.95 mL min  1 ). Eight experiments were evaluated with 3 factorsassigned to dummy factors, in triplicate and randomly. Threeresponses were evaluated: the area, retention time and width of the quercetin and kaempferol peaks. The analyses of the resultsand statistical interpretations of the effects were derived from theliterature [13]. The standard error was obtained from the dummyeffects. A response at the 5% level ( a ¼ 0.05) was consideredsignificant.  2.4.5. Hydrolysis and extraction Powdered  E. giganteum  (0.4 g of sample 2) was refluxed in awater bath at 90 7 1  1 C with 40 mL of various final molarconcentrations of HCl in 50% aqueous methanol for differenttime-periods (see next subsection). The liquid was filteredthrough cotton, and the residue was extracted twice with metha-nol (10 mL) under reflux (90 7 1  1 C) for 10 min. The extracts werecombined, and the methanol was removed at reduced pressure(30–35  1 C). Ten milliliters of water were added to the residue andextracted once with 20 mL and then three times with 10 mL of ethyl acetate. The combined ethyl acetate extracts were washedtwice with 50 mL of water and then filtered over 10 g of anhydrous sodium sulfate. The resulting solution was dried atreduced pressure ( o 40  1 C). The residue was resuspended in 5 mL of methanol, 5-fold diluted in the same solvent and filteredthrough a 0.45 m m PVDF filter (Millipore, MA, USA) prior to LCanalysis.  2.4.6. Experimental design and optimization of hydrolysisby response surface methodology (RSM) The acid hydrolysis of the  E. giganteum  raw material wasoptimized using central composite rotational design (CCRD) andresponse surface analysis [14,15] to maximize its efficiency while avoiding the degradation of the flavonoid aglycones.According to results obtained in preliminary research on theefficiency of the acid hydrolysis of flavonoid glycosides, the mostrelevant variables were identified as the HCl concentration andhydrolysis time. CCRD was developed using Minitab s 15.0 StatisticalSoftware (Minitab Inc., USA). Twelve experiments were performed intwo orthogonal blocks with 2 center points per block. The values of the variables were coded as  7 1 for the factorial points, 0 for thecenter points and  7 1.4142 for the axial points. The ranges of thevariables evaluated were 1.02 to 4.98M for HCl concentration and35.1 to 204.9min for hydrolysis time (see Table 4).The experimental data were fitted in the second-order poly-nomial model encoded in Y  ¼ b 0 þ b 1  X  1 þ b 2  X  2 þ b 11  X  21 þ b 22  X  22 þ b 12  X  1  X  2  ð 1 Þ where  Y   is the response variable to be modeled;  b 0 ,  b 1 ,  b 2 ,  b 11 ,  b 22 and  b 12  are the regression coefficients;  X  1  is the molar concentra-tion of HCl; and  X  2  is the hydrolysis time.The suitability of the model for each extracted flavonoid aglyconewas verified by analysis of variance (ANOVA). The optimum HClconcentration and hydrolysis time were obtained from the model byinspecting the response surface contour plots and using the Minitab s optimizer. The determination of flavonoid aglycones was carried outby LC–UV after hydrolysis at the optimum conditions, and the resultwas compared to the predicted value.  2.5. Statistical analysis Analyses were performed in triplicate. The individual data weregrouped after each experiment. The mean with the respectivedeviation was used as a measurement of the central tendency anddispersion (RSD, %). Microsoft Excel software (Microsoft, USA) andMinitab s 15.0 Statistical Software (Minitab Inc., USA) were employedfor ANOVA. 3. Results and discussion  3.1. Qualitative analysis The LC-DAD and LC–ESI-MS profiles of   E. giganteum  hydro-ethanolic extract are shown in Fig. 1, and the chromatographic,UV, MS and MS/MS data can be observed in Table 1.The molecular mass of the compounds was obtained fromtheir positive and negative ion electrospray mass spectra(ESI-MS), which described the corresponding protonated anddeprotonated pseudomolecular ions as well as the sodium andpotassium adduct ions (Table 1). In some cases, sodium formateadduct ions ([M þ 68]  and [M þ 136]  ), formed due to thepresence of formic acid in mobile phase A (data not shown) wereobserved in the negative ion mode ESI-MS. After the pseudomo-lecular ion identification, it was subjected to various CID energies(10, 20, 30, 50 and 70 V) in negative ion mode ESI-MS/MS todetect the site of substitution in the aglycones, thereby providingfirm evidence for the proposed compound structure.Thus, the structure of 12 compounds present in hydroethano-lic extract of   E. giganteum  were fully or partially characterizedusing the combined interpretation of the retention time, UVspectra and fragmentation patterns of the compounds obtainedby LC–DAD and LC–ESI-MS/MS. These data were compared with L.N. Francescato et al. / Talanta 105 (2013) 192–203 194  literature data, mainly those for the phenolics present in other Equisetum  species [7,16–18].  3.1.1. Characterization of flavonoid derivatives In combination with the application of different CID energiesin negative ion mode ESI-MS/MS, the UV spectral data were usedto characterize the site of substitution in the flavonoid aglycone.Band II is important for the identification of hydroxyl or methoxylsubstituents in the ring B, and hypsochromic shifts in Band I and/or II can indicate the methylation or glycosidation of the hydroxylgroups of flavonols [19]. Shoulders in the UV spectra can alsoindicate substitution in aglycones: kaempferol (266, 294sh,349 nm for 3-glycosides and 266, 318sh, 349 nm for 3,7-diglyco-sides) and quercetin (255, 266sh, 355 nm for 3-glycosides and255, 266sh, 294sh, 354 nm for 3,7-glycosides) [20].Four peaks (compounds 2, 6, 10 and 12) had UV spectracompatible with kaempferol glycosides. The UV spectra of com-pounds 2 and 6 showed a Band I maximum at 346.5nm and ashoulder at 318nm, indicating 3,7- O  substitution. In contrast, the UVof the compounds 10 and 12 showed a Band I maximum at 344.1nmand a shoulder at 290nm, indicating a 3- O  substitution [19,20]. The Y  07  [M-H-162]  at  m/z   609 (compound 2, [M-H]  of   m/z  771.1amu)and m/z   447 (compound 6,[M-H]  of   m/z  609amu)werethe base peak at 20 and 30V of CID energy, indicating the loss of aglucose residue at the 7- O  position, which is preferential [20]. Forcompound 2 (at 30V), the ion  0,2 X 0  [M-H-120]  at  m/z   651.2, withlow intensity (0.6%), and the ion Y  70 Z 3  1  [M-H-342]  at  m/z   428.7(1.6%) (Table 1) indicated the 1 - 2 interglucosidic linkage andbreakdown of a sugar moiety with the loss of one glucose residue,respectively, which are characteristic of the flavonoid 3- O -sophoro-side.The ion m/z  445.8indicatedtheflavonoid3-or7- O -glucosyl,andthe ion with  m/z   284.8 indicated the aglycone, kaempferol [21]. Forcompound 6, the characteristic ion Z 1  [M-H-180]  , indicating 1 - 2interglycosidic linkage, was not detected. The ions  0,2 X 0  [M-H-120]  at  m/z   488.9 and [M-H-282]  at  m/z   327 indicated breakdown withpartial loss of a hexose residue. The Y  3,7  0  [M-H-324]  ion at  m/z   284indicated kaempferol [21]. The homolytic cleavage of the 3,7- O -glycosidic bond in compounds 2 (at 70V) and 6 (at 50V) producedthe radical aglycone ions [Y  0 -2H]   m/z   283 (base peak) and[Y  0 -H]   m/z   284 as well as the aglycone [Y  0 ]  ion  m/z   284.9/ 285,indicatingadi- O -glycoside[22,23].Theions m/z  255/254.8and226.8/226.6, for compound 2 and 6, respectively, confirmed kaempferol[24], whereas the ion  m/z   151.2/150.6 indicated the presence of a7- O -glucoside [25]. Thus, compound 2 was identified as kaempferol-3- O -sophoroside-7- O -glucoside and compound 6 as kaempferol-3,7-di- O -glucoside. The influence of increasing fragmentation energy(CID) on the fragmentation pattern of compound 2 can be visualizedin Table 1 and Fig. 2. For compound 10 ([M-H]  of   m/z   609 amu), the base peak Y  03  [M-H-325]  at 30 V indicated the loss of a diglucose residue andthe kaempferol deprotoned aglycone at  m/z   284. The character-istic ion Z 1  [M-H-180]  at  m/z   428.9 indicated the 1 - 2 inter-glycosidic linkage of the flavonoid sophoroside. For compound 12([M-H]  of   m/z   447 amu), the Y  03  [M-H-163]  (base peak at30 V) indicated the loss of a glucose residue and kaempferol at m/z   283.9. The ion  0,2 X 0  [M-H-120]  , indicating the loss of asugar residue, was only detected at 20 V [21]. For compounds 10and 12, the homolytic cleavage of the 3- O -glycosidic bondproduced more intense radical aglycone [Y  0 -H]   ions  m/z   284and 283.9 than the aglycone [Y  0 ]  ions  m/z   284.9 and 284.8,respectively, confirming the glycosylation site at 3- O  position[22,23]. The ions  m/z   255 and 227, formed in MS/MS at 50 and70 V for compound 10 and 30 and 50 V for compound 12,respectively, are characteristic of kaempferol [24]. The ion  m/z  151, characteristic of kaempferol-7- O -glucoside, was not detectedfor either compound [25]. Compound 10 was identified askaempferol-3- O -sophoroside, and compound 12 was unambigu-ously identified as astragalin (kaempferol-3- O -glucoside) basedon a comparison of the retention times and MS and MS/MSspectra with those of a reference compound.Compound 1 and 4 exhibited UV spectra characteristic of aquercetin derivative. The Band I maximum at 351.3 and 348.9nmand the shoulder at 294nm of compound 1 and 4 indicated a 3,7- O substitution. The characteristic ions Z 1  and  0,2 X 0  of 1 - 2 intergly-cosidic linkages were not detected [21] for either flavonoid. Thepresence of ions at  m/z   300.5 and 300.8 at 30V, in addition to thefragment ions at  m/z   271 and 270.9 at 70V for compound 1 and 4,respectively, indicated the aglycone quercetin [24].The fragmentation pattern of compound 1 ([M-H]  of   m/z  787 amu) indicated the presence of 3 hexoses linked tothe aglycone by two or three  O -glycosidic linkages. At 30 V, the[M-H]  ion  m/z   787 was the base peak, followed by the[M-H-162]  ion  m/z   625 (34.2% R.A.), [M-H-325]  ion  m/z   462(30.7%) and [M-H-487]  ion  m/z   300.5 (3.7%), making it difficultto infer the location of the substitution from the relative abun-dances. At 50 V, the base peak was the ion  m/z   462, indicating thatthere is a hexose residue possibly linked at 3- O  position (the Fig. 1.  LC–DAD chromatogram at 254 nm (a) and LC–ESI-MS (negative ion mode) total ion current (TIC) chromatogram (b) of the hydroethanolic extract of   E. giganteum .The peaks are labeled according to the compounds listed in Table 1. L.N. Francescato et al. / Talanta 105 (2013) 192–203  195   Table 1 Retention time ( R t  ), UV absorptions ( l max ), negative and positive ion mode ESI-MS and negative ion mode ESI–MS/MS data of phenolic compounds presents inhydroethanolic extract of   E. giganteum . Compound  R  t   (min),LC–DAD(RSD , % ) a UV   k max  (nm) b Negative ion mode ( m/z  ) Positive ionmode ( m/z  )PhenoliccompoundMS[M-H]  CID(V)MS/MS (R.A.  % ) [M þ H] þ [M þ Na] þ [M þ K] þ 1 8.3 (0.84) 253.4,267sh,294sh,351.3787 10 787.1 (100), 625.1 (0.7) 789.1 811.3 Quercetin-tri- O -hexoside20 787.1 (100), 624.7 (5.9) 827.230 787 (100), 625 (34.2), 462 (30.7), 300.5 (3.7), 242.8 (9)50 462 (100), 300 (31), 298.8 (87.8)70 299.7 (2.7), 298.9 (100), 271 (16.4), 150.6 (2.1)2 10.1 (0.32) 265.2,318sh,346.5771.1 10 771.1 (100), 609.1 (59.3), 607.9 (0.4), 284 (0.4) 773.3 795.2 Kaempferol-3- O -sophoroside-7- O -glucoside20 771.1 (13.6), 609.2 (100), 284.8 (0.2), 284.1 (0.4) 811.230 770.7 (0.4), 651.2 (0.6), 609 (100), 445.8 (3.1), 428.7 (1.6),284.8 (2.5), 284 (3.6), 282.3 (0.4), 178.6 (0.3)50 609.1 (7.9), 446 (19.4), 428.9 (5.8), 325.9 (2.2), 285 (42.5),284 (100), 282.9 (43.1), 254.8 (5.3), 179.1 (1.8), 178.6 (1.4),151 (2.4)70 309.1 (0.6), 284.9 (29.5), 284 (49.9), 283 (100), 256.9 (1.7),255.9 (1.7), 255 (61.2), 226.8 (12.9), 181.7 (0.8), 151.2 (1.9)3 12.5 (0.30) 253.4,268sh,361.8 c 585.1 10 585.1 (100), 422.9 (4.2) 587.2 609.2 3-Hydroxyhispidin-3,4 0 -di- O -glucoside20 585.0 (100), 423.1 (21.2), 422 (2.8), 259.8 (2.5), 259.1 (1.9),241 (2), 217.2 (2.7), 215.9 (2.2), 202.8 (2.4)625.230 585 (63.6), 423.1 (100), 422.1 (26.7), 379.1 (14.1), 259.9(90.3), 259 (96.4), 257.8 (6.5), 230.8 (28), 217 (41.7), 216(44.8), 214.9 (6.3), 202.9 (52.3), 159 (6.9)50 259.5 (10.1), 259 (8.4), 216.9 (11.5), 215.9 (10.6), 202.9(100), 188 (7.6), 186.7 (19), 174.3 (7.8), 171 (5.1), 159 (60.2),142.4 (6.4)70 203 (19.9), 186.9 (6.9), 175.3 (3.7), 172.8 (3.2), 170.8 (8.9),159 (100)4 13.9 (0.66) 253.4,267sh,294sh,348.9625.1 10 625 (100), 462.9 (6.1) 627.3 649.2 Quercetin-3,7-di- O -glucoside20 625.1 (100), 462.8 (31.3), 461.8 (4.6) 665.130 624.5 (6.9), 463 (31.5), 462 (100), 300.8 (29.3), 299 (11.7)50 301 (10.3), 299.9 (3.9), 298.8 (100), 270.9 (3.2)70 300.8 (10.7), 298.6 (48.6), 270.9 (100), 243.1 (4.1), 242.2(5.3)5 16.5 (0.73) 246.2,295sh,327.3625.1 10 624.9 (100), 462.9 (42.4) 627.1 649.2 Quercetin-3- O -(caffeoyl)-glucoside20 624.8 (10.3), 463.1 (100), 462.4 (42.1), 341.5 (13.5) 665.030 463.1 (100)50 300.8 (46.8), 300.1 (72.5), 270.9 (100), 255.1 (99), 227.1(30.4)70 270.6 (93.3), 254.7 (84.1), 242.6 (100)6 18.2 (0.85) 265.2,318sh,346.5609 10 609 (100), 489.2 (0.6), 488.8 (0.5), 447 (12.4), 446 (1.1), 285(0.4), 282.9 (0.5)611.2 633.2 Kaempferol-3,7-di- O -glucoside649.220 609.1 (65.2), 489.1 (6.8), 447 (100), 445.8 (9.4), 285.1 (23.4),282.8 (1.5)30 609.2 (1.6), 488.9 (2.3), 460.1 (1.1), 447 (100), 446 (77). 327 (4),285 (85.8), 283.9 (3.6), 283 (24), 269.2 (1), 255.1 (2), 151 (0.9)50 446.7 (0.7), 445.9 (0.5), 297.2 (0.6), 285 (60.5), 284 (11), 283(100), 254.8 (13.8), 226.6 (2.2), 150.6 (1.1)70 284.9 (18.5), 282.9 (44.3), 254.9 (100), 227.1 (10.2), 183 (5.3)7 22.2 (0.64) 267.6,353.5625 10 625.1 (100) 627.2 649.2 Flavonol-di- O -hexoside20 625.2 (58.1), 624.6 (100), 463.2 (8.1) 665.230 625.1(34.5),624.6(35),463(100),462.2(19.4),461.5(7),299.9(5.6)50 300.4 (22.5), 299.9 (18.3), 299 (64.2), 271.1 (100), 254.8 (25.6),201.1 (10.4)70 270.7 (84.2), 255.2 (100), 254.6 (95.2), 227 (21.1)8 23 (0.73) 253.4,272sh,371.4423 10 423 (100), 379.1 (1.8), 378.4 (0.8), 286.9 (1.2), 261 (8.6), 260(1.5), 217 (1.1), 215.9 (0.8)425.1 447.1 Equisetumpyrone463.120 423 (100), 261.1 (62.7), 260.1 (49.2), 216.9 (32.3), 216 (25.5),202.9 (49.5), 198.8 (8), 189.2 (8), 188.1 (17), 172.9 (7.6), 161(6.9), 159 (4.9), 135 (8.8), 127.1 (5.6)30 260.9 (14.4),259.7 (12.5), 216.9 (24.7), 216.1 (6.1), 215.5(10.7), 202.8 (100), 197.3 (6.7), 188 (18.7), 187 (13.1), 174.1(6.1), 172.8 (11.2), 172.1 (6.7), 161.1 (7.3), 159 (22.4), 135.1(14)50 202.8 (100), 186.9 (34.4), 175 (7.3), 171.1 (4.4), 159 (41), 158(4.9), 144 (7.8), 135 (15.5), 108.9 (7.3)70 203 (36.6), 186.7 (18.6), 160.8 (11.8), 159.8 (5.5), 159 (100),135.1 (11.6), 134.3 (12.1), 132.6 (7.3) L.N. Francescato et al. / Talanta 105 (2013) 192–203 196
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