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A fragmentation study of dihydroquercetin using triple quadrupole mass spectrometry and its application for identification of dihydroflavonols in Citrus juices

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A fragmentation study of dihydroquercetin using triple quadrupole mass spectrometry and its application for identification of dihydroflavonols in Citrus juices
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  A fragmentation study of dihydroquercetin using triplequadrupole mass spectrometry and its application for identification of dihydroflavonols in  Citrus  juices Beatriz Abad-Garcı´ a, Sergio Garmo´ n-Lobato, Luis A. Berrueta, Blanca Gallo*and Francisca Vicente Departamento de Quı´mica Analı´tica, Facultad de Ciencia y Tecnologı´a, Universidad del Paı´s Vasco/Euskal Herriko Unibertsitatea, P.O. Box 644,48080 Bilbao, Spain Received 20 February 2009; Revised 23 June 2009; Accepted 27 June 2009 A mass spectrometric method using electrospray ionization with triple quadrupole and quadrupoletime-of-flighthybrid(Q-Tof)massspectrometryhasbeenappliedtothestructuralcharacterizationofdihydroflavonols. This family of compounds has been studied by liquid chromatography/tandemmassspectrometry (LC/MS/MS)for thefirst time inthis work.A comprehensive studyofthe production MS spectra of the [M R H] R ion of a commercially available standard has been performed. Themost useful fragmentations in terms of structural identification are those that involve cleavage of theC-ring, resulting in diagnostic ions of dihydroflavonol family:  1,3 A R 0  ,  1,2 B R 0  ,  1,2 B R 0  -CO,  0,2 A R 0  ,  0,2 A R 0  -H 2 O, 0,2 A R 0  -CO,and 0,2 A R 0  -H 2 O-CO,thatallowthecharacterizationofthesubstituentsintheA-andB-rings. In addition to those ions, other product ions due to losses of H 2 O and CO molecules fromthe Y R 0  ion were observed. Their fragmentation mechanisms and ion structures have been proposed.The established fragmentation patterns have been used to successfully identity three dihydrofla-vonols found in tangerine juices for the first time. Copyright # 2009 John Wiley & Sons, Ltd. Polyphenols, widely distributed in fruits and vegetables,have a great importance in human diet as they have well-recognized physiological actions and are the object of numerous medical studies about their beneficial properties. 1 Moreover, phenolic compounds have an important role inthe nutritional, organoleptic and commercial properties of agricultural foodstuffs, since they contribute to their sensorypropertiessuch ascolor, astringency, bitternessandflavor. 2,3 Due to the importance of flavonoids as contributors of  beneficial health effects and their importance in the qualityproperties of fruits and vegetables, the identification and/orstructural determination of such compounds occurring inplant tissues or other biological systems play important rolesin many areas of science. However, despite the extensiveresearch performed in this field, many compounds stillremain unidentified in fruits and vegetables.Several publications describe advances in  Citrus  flavonoiddetermination, especially by high-performance liquidchromatography (HPLC), usually in combination withdiode-array detection for their identification and character-ization. 4,5 However, in recent years, mass spectrometry (MS)hasbecomethemostpopularandmostimportantmethodfordetection offlavonoids. Nowadays,liquidchromatography/tandem mass spectrometry (LC/MS/MS) is the mostconvenient technique for on-line characterization, thanksto its superior sensitivity, high selectivity and resolutionpower which allow direct screening of natural products,avoiding the previous need for laborious isolation of polyphenols. MS fragmentations of flavonoids and theirglycosides have been comprehensively studied and theirfragmentation patterns have been extensively applied fortheir characterization in natural sources in the literature. 6,7 Several papers have already described the fragmentationofthemajority of  Citrus flavonoids, classifiedinto threemainfamilies (flavanones, flavones, and flavonols), 8 which occurprincipally in the peel. Here we report another family of flavonoids, dihydroflavonols, less abundant in  Citrus  fruitand not studied until now by LC/electrospray ionization(ESI)-MS/MS. To the best of our knowledge only one workdescribes briefly the fragmentation pattern of dihydroflavo-nols by MS. These authors used gas chromatography(GC)/MS using electron ionization (EI) as ionization mode, 9 inwhich mass fragmentations are completely different fromthose in ESI mode. However, because flavonoids arethermolabile and suffer degradation at high temperatures,LC/MS is more suitable than GG/MS techniques for theiranalysis. In this paper, the fragmentation pathways of dihydroflavonolshavebeenstudiedbyLC/ESI-MS/MS,andthe established fragmentation patterns have been used forsuccessful identification of three dihydroflavonols in tanger-ine juices for the first time. RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom.  2009;  23 : 2785–2792Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.4182 * Correspondence to :B.Gallo,DepartamentodeQuı´micaAnalı´tica,Facultad de Ciencia y Tecnologı´a, Universidad del Paı´s Vasco/EuskalHerrikoUnibertsitatea,P.O.Box644,48080Bilbao,Spain.E-mail: blanca.gallo@ehu.esContract/grant sponsor: Universidad del Paı´s Vasco/EuskalHerriko Unibertsitatea; contract/grant number: 13468/2001and 15978/2004. Copyright # 2009 John Wiley & Sons, Ltd.  EXPERIMENTALReagents, solvents and standard phenolics Methanol (Romil, Chemical Ltd., Heidelberg, Germany) wasof HPLC grade. Water was purified on a Milli-Q system(Millipore, Bedford, MA, USA). Glacial acetic acid, ascorbicacid and sodium fluoride provided by Merck (Darmstadt,Germany) were of analytical quality. Standard of thedihydroquercetin (( þ )-taxifolin) (Red Label quality) waspurchased from Extrasynthe`se (Genay, France). Standardstock solution was prepared in methanol and dilutions weremade in the initial mobile phase. The other dihydroflavonolsstudied were analyzed from  Citrus  juice that was laboratory-made from 1kg of tangerines ( Citrus reticulate , Spanishcultivar: Clemenule). A volume of 50mL of an aqueoussolution of NaF (1mg/L) was added to the juice in order toprevent the activity of polyphenoloxidase (PPO). The juicewascentrifugedat6000rpmfor15minat4 8 C.A1mLaliquotof this juice was freeze-dried for preservation and extractedat the time of analysis with 2mL of a mixture of methanol/water/acetic acid (30:69:1, v/v/v) with ascorbic acid aspreservative (2g/L). Mixing was carried out by vortex andtheextractionwasperformedinanultrasonicbathfor15minat room temperature. The extract was centrifuged at4000rpm for 4min and passed through a 0.45 m m PTFEfilter (Waters, Milford, USA) prior to its injection into thechromatographic system. 10 HPLC analysis The HPLC system was an Alliance 2695 (Waters, Milford,USA)coupledtoaWaters2996DAD.Areversed-phaseLunaC18(2) column (150  4.6mm i.d. and particle size 3 m m;Phenomenex, Torrance, USA), with a Nova-Pack C18 guardcolumn (10  3.9mm i.d., 4 m m; Waters) was used. The flowrate and column temperature were set to 0.8mL/min and30 8 C, respectively.A gradient program for general polyphenol analysis 10 wasemployed: the eluents were acetic acid/water (0.5:99.5, v/v)(phase A) and methanol (phase B); initially 0% B for 2min, alinear gradient to 15% B at 6min, held isocratic until 12min,linear gradient to 20% B at 15min, 20% B constant until35min, linear up to 35% B at 90min, 35% B constant until136min, and finally linear gradient to 0% B at 145min.A 10 m L volume of standard solution (about 50 m g/L) wasinjected. The chromatograms were monitored at 280nm andcomplete spectral data were accumulated in the range 250–600nm each second. Triple quadrupole mass spectrometry Mass spectra were obtained using a Quattro-micro triplequadrupole mass spectrometer (Micromass, Milford, MA,USA) coupled to the exit of the DAD and equipped with a Z-spray ESI source. A flow of 70 m L/min from the DAD eluentwas directed to the ESI interface using a flow splitter.Nitrogen was used as desolvation gas, at 300 8 C and a flowrate of 450L/h, and no cone gas was used. A potential of 3.2kV was used on the capillary for positive ion mode and2.6kV for negative ion mode. The source block temperaturewas held constant at 120 8 C.MS 1 spectra, within the  m/z  range 50–1000, were acquiredat different cone voltages (15, 30 and 45V) in the positivemode and at 30V in the negative mode. MS 2 product ionspectra were recorded using argon as collision gas at 15  10  3 mbar and different collision energies in the range 5–40eV. The resolution of the quadrupole mass analyzerscorresponded to peak widths of less than 1 m/z  unit at the baseline, and a transmission window of approximately oneunit (parameter value 14) was selected in the first quadru-pole. 11 Q-Tof mass spectrometry Medium-resolution mass spectra were obtained using a Q-Tof QSTAR Elite mass spectrometer (Applied Biosystems,CA, USA) coupled to a Harvard syringe pump. A solution of 60 m g/mL of dihydroquercetin standard in MeOH/H 2 O/AcOH/acetonitrile (25:37.5:0.04:37.5) was injected at a flowrateof25 m L/minintoaTurboIonsprayionsourceinpositivemode.Gasparameters(N 2 )were:nebulization20,curtain20,CAD 5. A potential of 5.5kV was used on the capillary.Product ion spectra of the ion at  m/z  305 were acquired, withaccumulation time 1.0 s. Tof parameters were: pulsefrequency 11.038 KHz, pulse duration 9 m s. Spectra wererecorded from  m/z  60 to 700. Calibration of the Q-Tof withreserpine was performed. MS 2 product ion spectra of the ionat  m/z  305 were recorded at two collision energies: 20 and30eV. Mass resolution of the Q-Tof is 8000 FWHM and thetransmission window in the first quadrupole was 1 m/z  unit. Computational methods Marvin and Calculator Plugins (Marvin version 5.2.0, 2009,ChemAxon 12 ) were used for drawing, tautomer generationand initial structure calculation using molecular mechanicsmodels. Final most stable structures, heats of formation andpartialatomicchargeswerethencalculatedusingSpartan’04(Wavefunction, Irvine, CA, USA) using a density functionaltheory (DFT) B3LYP model with a parametric set 6.31G  . Toassess if a structure in a reaction is more plausible thananother, its  D H f   has been considered as the main criterion.For every structure proposed in the schemes, severalreasonable candidate structures have been proposed andcomputed (see Supporting Information). Nomenclature and abbreviations The nomenclature proposed by Domon and Costello 13 forglycoconjugates was adopted to denote the fragment ions(Scheme 1). Ions containing the aglycone are labeled  k,l X  j , Y  j and Z  j , where  j  is the number of the interglycosidic bond broken, counting from the aglycone, and the superscripts  k and  l  indicate the cleavages within the carbohydrate rings.Theglycosidicbondlinkingtheglycanparttotheaglyconeisnumbered 0.The flavonoid aglycone fragment ions can be identifiedaccording to the nomenclature proposed by Ma  et al . 14 (Scheme 2). For free aglycones, the  i,j A þ and  i,j B þ labels referto the fragments containing intact A- and B-rings, respect-ively, in which the superscripts  i  and  j  indicate the C-ring bonds that have been broken. For conjugated aglycones anadditionalsubscript0totherightoftheletterisusedtoavoidconfusion with the A þ i  and B þ i  ( i   1) labels that have been Copyright # 2009 John Wiley & Sons, Ltd.  Rapid Commun. Mass Spectrom.  2009;  23 : 2785–2792DOI: 10.1002/rcm 2786 B. Abad-Garcı´a  et al.  used to designate carbohydrate fragments containing aterminal (non-reducing) sugar unit. 13 RESULTS AND DISCUSSIONOverview of UV and single-stage MS spectra ofdihydroflavonols It is extensively known that diode-array detection is aninvaluable tool for the tentative identification of mainphenolic structures since each class exhibits a characteristicUV-visible spectrum; however, dihydroflavonols cannot bedifferentiated from other flavanones using only their UV-visible spectra (both present the absorption band IIdetermined by the A-ring at 285nm and the absorption band I determined by B-ring as a plateau at 310–350nm).Generally, dihydroflavonols are more polar and elute beforethe flavanones, but glycosylation patterns can modifysignificantly their retention times. Therefore, becausecommercial standards of the full range of dihydroflavonolsare not available, their identification is not possible usingonly a HPLC-DAD method.Massspectrometryallowsdirectscreeningofthesenaturalproducts, providing structural information about them andremoving the previous need for laborious isolation of polyphenols. Each flavonoid aglycone family presents acharacteristic MS fragmentation pattern that allows itsidentification. 11 For determination of this pattern fordihydroflavonols, single-stage MS spectra and ESI( þ )-CID-MS/MS product ion spectra of [M þ H] þ of the dihydro-flavonol standard (dihydroquercetin) were recorded (Fig. 1)and its fragmentation pattern was proposed (Scheme 2).Little fragmentation was observed in MS 1 spectra (fullscan) at a conevoltage of 15V. [M þ H] þ or [M–H]  ions werethe base peaks in positive and negative modes, respectively.Intense adducts with the sodium ion (present in the mobilephase) are also observed in ESI( þ ), and with sodium acetate[M-H þ AcONa]  or with sodium acetate plus methanol [M–H þ AcONa þ MeOH]  in ESI(–). Clusters [2M–H]  and[2M þ Na–H]  from the mobile phase were also detected inESI(–). These adducts and molecular complexes give extracertainty to the molecular mass determination. Fragmentations of dihydroquercetin aglycone The product ion mass spectrum of dihydroquercetin ( m/z 305) is shown in Fig. 1. The most useful fragmentations intermsofdihydroflavonolidentificationarethosethatinvolvecleavageoftheC-ringatpositions1/3,1/2and0/2,resultingin structurally informative  i,j A þ and  i,j B þ ions (Scheme 2).These three fragmentation routes are designated as Scheme 2.  Structure and fragmentation of the dihydroflavo-nol dihydroquercetin. C-ring bonds labelling of flavonoidskeleton. Scheme 1.  General MS fragmentations of flavonoid glyco-sides. Atom labelling of flavonoid skeleton. Figure 1.  MS 2 productionspectraof[M þ H] þ ofdihydroquercetinstandard(conevoltage15V, collision energy 35eV). Copyright # 2009 John Wiley & Sons, Ltd.  Rapid Commun. Mass Spectrom.  2009;  23 : 2785–2792DOI: 10.1002/rcm Fragmentation study of dihydroquercetin 2787  pathwaysI,IIandIII,respectively.PathwayIisequivalenttothat reported previously by Claeys and co-workers forflavone and flavonol aglycones. 14 PathwayIgivesrisetothe 1,3 A þ ionat m/z 153,whichlosesfurther successive CO molecules to provide the fragmentions at  m/z  125 and 97 (Scheme 3). Losses of CO moleculesfrom the  1,3 A þ ion have been also reported for kaempferolflavonol by March and co-workers 15 using a quadrupoletime-of-flight mass spectrometer with an ESI source operat-ing in negative mode.Pathway II resulted in the  1,2 B þ ion at  m/z  123 (Scheme 4).This rupture was also observed for flavan-3-ols andflavonols; therefore, the  1,2 B þ ion is a diagnostic ion of flavonoids with a hydroxyl group at the 3-position. Theoccurrence of a 3-OH group in the C-ring seems to assist thefragmentation of the bond between O-1 and C-2. 16 Thisfragmentprovidesinformationaboutthenumberandtypeof substituents of the B-ring. The mechanism proposed toexplain this fragmentation suggests a rearrangement of theC-ring which is favored by the formation of a stable benzyliccation.Pathway III leads to the  0,2 A þ ,  0,2 A þ -H 2 O,  0,2 A þ -CO and 0,2 A þ -H 2 O-CO product ions at  m/z  167, 149, 139 and 121, bylossesofaCOandaH 2 Omolecule.ThelossofCOfrom 0,2 A þ can imply an internal rearrangement. Unlike flavonols,the formation of a new conjugated double bond at C3–C4favors the loss of the hydroxyl group at the 3-position,giving rise to a  0,2 A þ -H 2 O ion at  m/z  149. The mechanismproposed to explain this rupture is shown in Scheme 5, inwhich a dehydration and subsequent rupture between bonds 0 and 2 take place (this should imply a hydrogenmigration).In this way, Dekoster and co-workers found two ions at m/z  165 and 137 in the study of kaempferol aglyconefragmentations, which were assigned to  0,2 A þ and  0,2 A þ -COfragment ions, respectively. 17 These ions correlate well withthe ions at  m/z  167 and 139 of dihydroquercetin, consideringthe two additional hydrogen atoms in the C-ring. Therefore,a similar mechanism can be proposed for this aglycone. Inaddition, the lack of a double bond in the C-ring incomparison with kaempferol aglycone allows the priorloss of water. Thus, it is reasonable to propose that the 1,2-diketobutene structure proposed by Koster and co-workersfor kaempferol should be generated as shown in Scheme 5.In addition to the  i,j A þ and  i,j B þ ions already discussed,productionsduetolossesofH 2 OandCOmoleculesfromthe[M þ H] þ ion were also observed. The loss of the 3-hydroxylgroupwouldbefavoredbytheformationofadoublebondinthe C-ring. Finally, the losses of CO molecules are attributedto phenolic groups and the keto function at the 4-position(Scheme 6).All fragment assignments have been supported usingaccurate mass measurement data obtained using the Q-Tof.Accurate  m/z  values of product ions of [M þ H] þ found for Scheme 3.  Mechanism proposed for the formation of  1,3 A þ and  1,3 A þ -nCOproduct ions in the case of the protonated dihydroquercetin dihydroflavonol. Scheme 4.  Mechanism proposed for the formation of  1,2 B þ 0  and  1,2 B þ 0 -COproduct ions in the case of the protonated dihydroquercetin dihydroflavonol. Copyright # 2009 John Wiley & Sons, Ltd.  Rapid Commun. Mass Spectrom.  2009;  23 : 2785–2792DOI: 10.1002/rcm 2788 B. Abad-Garcı´a  et al.  dihydroquercetin correlate with the expected values calcu-lated using molecular formulae within the instrumentalprecision, as shown in Table 1. Examples of characterization of dihydroxy-flavonols in extracts of tangerine juice byLC/DAD-ESI-MS/MS In tangerine juice of the Clemenule variety, three flavonoidswith flavanone-like UV-Vis spectra have been detected atretention times of 21.2, 27.1 and 36.2min under ourchromatographic conditions (Fig. 2). Their early retentiontimes suggest their identification as dihydroflavonols.MS 1 spectra in positive mode at a cone voltage of 15Vshow the [M þ H] þ ions at  m/z  613, 597 and 627, respectively,together with their sodium ion adducts. The MS 1 spectraobtained in negative mode at a cone voltage of 30Vcorroborated the identity of [M–H]  ions based on theadducts with bisulfate [M þ HSO 4 ]  of great intensity (HSO  4 is an impurity of the mobile phase derived from thecommercial acetic acid).In order to determine their glycosylation patterns, MS 2 product ion spectra of [M þ H] þ at a collision energy of 10eVwere obtained following the stated guidelines established inapreviouswork. 18 Thesespectraarealmostidenticaltothose Scheme 5.  Mechanism proposed for the formation of  0,2 A þ 0  and  0,2 A þ 0 -COand  0,2 A þ 0 -H 2 O and  0,2 A þ 0 -H 2 O-CO product ions in the case of the protonateddihydroquercetin dihydroflavonol. Scheme 6.  Mechanism proposed for the formation of [M þ H þ –H 2 O] þ , [M þ H þ –CO] þ and [M þ H þ –H 2 O–CO] þ product ions in the case of the protonated dihydroquercetindihydroflavonol. Copyright # 2009 John Wiley & Sons, Ltd.  Rapid Commun. Mass Spectrom.  2009;  23 : 2785–2792DOI: 10.1002/rcm Fragmentation study of dihydroquercetin 2789
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