CHARACTERIZATION OF C-GLYCOSIDIC FLAVONOIDS FROM BRAZILIAN SPECIES USING LC/MS EXACT MASS MEASUREMENT

1 2 2 NOTE M. McCullagh , C.A.M. Pereira , J.H. Yariwake 1Waters Corporation, Floats Road, Wythenshawe, Manchester, M23 9LZ, UK 2Universidade de São Paulo, Instituto de Química de São Carlos, São Carlos, SP, Brazil

OVERVIEW

This application note describes the analysis of extracts Recently issues have arisen with Kava Kava, a natural from Passiflora species using real time centroid exact health product, which is used for its anti-depressant and mass measurement. The system used comprised of an anti-anxiety properties. Investigations into the safety of LCT™ orthogonal acceleration (oa-TOF) time of flight Kava have taken place within several European mass spectrometer with LockSpray™ source, Waters® countries, including Germany, U.K., Switzerland, 2996 PDA and Waters Alliance® HT 2795 France, Spain and parts of Scandinavia. In Switzerland Separations Module. and Germany cases of liver damage were reported. In

Application the US the FDA has investigated Kava's safety, where INTRODUCTION as in Canada the public were advised not to take the

EU Directive 2001/83/EC will regulate and produce a supplement until product safety could be assured. The set of harmonized assessment criteria for the efficacy Kava market in Germany alone is $25m. Proving the and safety "Herbal Medicinal Products for traditional efficacy of such a product is economically viable. use". The current and future E.U. member states will Such issues illustrate how regulation currently affects produce and abide by this directive, making 28 states the natural health product market and also why new in total. ESCOP (European Scientific Cooperative on legislation is required. Phytotherapy) aim to influence the EU in order to change law, so that patent protection can apply to the properties of natural products discovered, as a result interest in this industry will grow.

Waters® LC/MS System, featuring the Alliance® HT 2795 Separations Module, 2996 Photodiode Array Detector, Micromass® LCT Mass Spectrometer (with LockSpray™) and MassLynx™ Software.

ApplicationNOTE also in South Brazil. studied dueto its utilizationinArgentinaand possibly (sedative/tranquilizing). are utilizedasphytomedicines Several performed. species isthe indicated bytheBrazilianPharmacopoeia (Passifloraceae) For thisstudyanalysisof Authentication ofplantspeciesand • the Molecularbiologicalassaystodetermine • • Standardizationofanalyticalmethodswithan • require: Trends andchallengesinPhytomedicineresearchwill necessity toprofileplantmakeup. environment inwhichitgrows,thereforethereisa A plantspeciesprofilecanbeaffectedbythe Stabilitystudies,degradationissuesrelatingto • Theextractionprocedureisablendingofextracts • Purityisanissue,heavymetalcontent, • Regulation assessment ofherbalmedicines. and assessingthemonographsproducedfor isconstantlyupdating The EuropeanPharmacopoeia frequently substitutedby Brazilian climatethis Howeverinthe sedative andtranquilizingproperties. juices). Additionally very well. Here an alternative to well.Hereanalternative very standardization ofphytochemicalscreening. mechanism ofaction. extracts, standardizeddoses. Controlled clinicaltrials,requiringstandardized TLC, CEandCEC. advance inusingHPLC-MS,UV, NMR,FT-IR, storage conditions,packaging,humidityetc. within afinalproductherbalformulation. to produceaknownquantityofactiveingredients products i.e.OTCherbalremedies. natural ecoli) haveallbeenfoundinimported fungi, mycotoxinsandbacteria(salmonella fumigants, pesticides,radioactivecompounds, Passiflora alata is widelyknowninEuropeduetoits , Passiflora (Passifloraceae) P Passiflora . caerulea and thishasbeenfoundtobe Passiflora incar Passiflora edulis Passiflora species doesnotgrow should alsobe species hasbeen nata L (utilized in . species Medicinal illustrated. Figure 3theLockSpraydualelectrospraysourceis schematic ofanOa-TOFisillustratedinFigure5.In acquisition ofrealtimeexactmasscentroiddata;a of thedata.Oa-TOFisusedasatoolforeasy an extradegreeofinfor spectra withhighmassaccuracy(<5ppm)providing is utilizedforqualitativestudies,generatingfull W available massrangeandioncollectionefficiency. analytical toolbecauseoftheacquisitionspeed, Oa-TOF-MS combinedwithHPLCisapowerful damage inthepatients. Kavalactones beingextracted,resultinginliver method usedmayhaveresultedinhighlevelsof activity. InthecaseofKavaextraction components maypossessextremelypotenttoxic minor componentsisessentialsince Specific dataisrequired,andtheabilitytodetect identify allcomponentsofanaturalhealthproduct. The issuesrelatingtoKavahighlighttheneed skeletal structure. have thesameluteolin-typeorapigenin-typeof due tothepresenceofotherflavonoids,whichalso the flavonoidsofinterestfromdegradationproducts has beenpossibletodistinguishandcorrectlyassign Thereforeit spectra acquisitionhasbeenperformed. phytomedicines. Inthisstudyoa-TOFLC/MSfull flavonoid isomersincomplexmixturessuchas are impor techniques combinedwithexactmassmeasurement derivatives; frequentlyoccurringasisomers).LC/MS mainly C-glycosylflavones(apigeninandluteolin ithin manylaboratoriesthehighdutycycleofTOF tant toolsforunequivocalidentificationof Passiflora 1,2,3. species containflavonoids, mation thataidsinterpretation

Figure 2. ZSpray source schematic.

The use of exact mass measurements, particularly for low molecular weight compounds, provides a greater confidence in compound confirmation through the ability to determine actual Figure 1. Dual electrospray source mounted on an elemental compositions. With the introduction of LockSpray as a LCT oa-TOF instrument. routine and rugged hardware configuration for the LCT, it is Oa-TOF LC/MS instrumentation can be utilized to speed up possible to provide a single reference peak against which any discovery of new natural products and to play a role in subsequently acquired mass spectra are accurately mass addressing many of the issues of this market, where regulation is measured. Historically it was common working practice on now being applied. The LCT™ incorporates ZSpray™ technology, magnetic sector and orthogonal-acceleration time of flight giving increased sensitivity with the ruggedness and robustness (oa-TOF) mass spectrometers to introduce a reference solution required for routine analysis of plant extracts. Dual orthogonal into the eluent stream prior to the ionization source, in order to sampling of the sample spray exiting the electrospray capillary allow exact mass measurement of LC peaks. The associated takes place: the spray is directed towards the baffle plate where inherent problems of teeing in the reference compound can collection of involatiles from sample matrices or mobile phase result in a variation in the signal intensity when LC gradients are additives takes place. Ions are extracted electrostatically through applied; mass interference with analytes have the same nominal 90 degrees and they enter through the sample cone. The ions mass and suppression of the reference response with high are further electrostatically bent through 90 degrees and enter concentration analyte response. In these instances mass through the sample extraction cone, where they enter the mass measurement errors can occur. spectrometer. The majority of the involatile ions that actually enter the ion block are pumped away. Due to the source design the sample cone orifice can be made larger to allow more ions to enter the ion block, hence providing greater sensitivity. Dual orthogonal spray sampling provides greater source robustness and sensitivity, and is the exact requirement for dealing with real samples. This produces a perfect combination for natural product analysis.

ApplicationNOTE Figure 3.SchematicoftheLockSpraysource. Highsensitivity—efficient • Elevatedmassspectralresolution>5000FWHM. • as follows: "Fingerprint" ofnaturalmedicinesisdescribed functionality thatcanbeutilizedtoprovidethe compared withthatofaquadrupoleinFigure4.The The resolutionthatcanbeachievedusingtheLCTis MS&In-sourceCID(CollisionInduced • 3orders ofmagnitudelinearity. • • • exactmassmeasurements(<5ppmRMS). • Elemental compositionoffragments • In-sourceCIDgeneratingpseudoMS/MS • Dissociation). Quantitationstudies. • Fullspectrumacquisition. • Lowlevelanalytedetection. • chromatograms). removedusingexactmass interferences Increased selectivity(nominalmassmatrix composition calculator). help toidentifyunknowns(elemental confidence inconfir determined. fragment ionswithelevatedMS resolution. 'non-scanning' ming targetanalytes instrument. 100 100 Figure 4.Oa-TOF/Quadrupoleresolutioncomparison. Figure 5.Schematicofoa-TOF. EXPERIMENTAL SAMPLES Passiflora STANDARDS % % 0 0 4 54647484950515253545556507 506 505 504 503 502 501 500 499 498 497 496 95 clean-up (Sep-Pak procedure) SPE Pharmacopoeia mL solventaccordingtoBrazilian ethanol-water (2:1v/v, 1gplant/10 Campinas -SP, Brazil) Campinas -SP, Brazil) and Sample pr P P. incarnata Passiflora alata V , ,isoorientinandrutin 60% methanol-H (4 mg/mL) analysis ofhydromethanolicfraction in RibeirãoPreto,SP, Brazil); . caerulea egetal material: 500 5 00 eparation: (supplied byIAC- , (suppliedbyCPQBA- 501 5 01 and ® dried leavesof 2 Quadrupole 1000 FWHM O) andLC-MS C Oa-TOF 5000 FWHM P. edulis 18 Extraction with 502 502 , elutionwith (grown ma ss

LC/MS CONDITIONS The flavonoids of interest for this study, , orientin,

® vitexin and are shown in Figure 6. Orientin and Column Symmetry C18 (250 mm x 4.6 mm vitexin are 8-C glycosides and respectively differ from 6-C x 5mm) with guard column (2 cm glycosides isovitexin and isoorientin through the position of the x 3.9 mm x 5 µm) - LC attached glucose group as illustrated. Isovitexin and isoorientin Column temperature 35°C differ with respect to the absence or presence of the hydroxy

Mobile phase ACN (B) : H2O (0.2% HCOOH) group shown. This also applies to vitexin and orientin. Orientin (A) Gradient: 0-10 min: 15% B; and isoorientin have the elemental composition C21H20O11, 10-40 min: 15-30% B; 40-50 whereas vitexin and isovitexin have an elemental composition min: 30-15% B of C21H20O10.

Flow 1 mL/min - split 1:4 Passiflora edulis 100 Instrument LC-TOF-MS, (Micromass®) LCT equipped with LockSpray dual- electrospray ion source Mode ESI positive and negative

ESI Voltage +VE = 3 kV -VE = 2.9kV % MS Sample cone voltage 20 V

Exact mass reference Leucine enkephalin, [M+H]+ = 556.2771 - [M-H] = 554.2615 0 Time 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 m/z range 100 to 800 m/z Figure 7. Passiflora edulis extract positive ion mode electrospray RESULTS total ion chromatogram.

OH As can be seen from Figure 7, analysis of the Passiflora edulis OH HO OH extract has resulted in a plethora of major and minor HO O O OH OH OH HO components being identified in one analysis. The resultant O HO O OH electrospray positive ion mode total ion chromatogram indicates OH O HO OH the complexity of the natural product extracts being identified. OH OH O The advantage of oa-TOF LC/MS is the ability to achieve full

Isoorientin Orientin spectra acquisition for such a large number of components in one analysis. For each Passiflora species extract the determination of the presence of isoorientin and orientin was OH

OH HO required. From extraction of the m/z 449 mass chromatogram OH

HO O Figure 8 was produced. O OH OH HO

O HO O

OH O HO OH

OH OH O

Isovitexin Vitexin

Figure 6. Flavonoids of interest characterized in Passiflora extracts.

ApplicationNOTE 449 masschromatogram. Figure 8.Passifloraedulisexpandedextractedm/z 1 A and B, was determined tobeC A andB,wasdetermined composition ofthemassspectrageneratedforpeaks In bothcases,themostprobableelemental with asetofdefinedparameterscanbedeter chromatogram obtainedfor Figure 8forthem/z449extractedmass peaks arelabelledA,B,CandDasshownin m/z 449weredetected.Thefourmostabundant concluded thatAandBareisoorientinorientin. exact massmeasurementsobtaineditcanbe elemental compositionC generated forpeakAofFigure8showsthatthe Figure 9theexactmassandelementalcomposition of C molecular ionspecieswithanelementalcomposition isoorientin wasm/z449.1084,fortheprotonated The targetflavonoidmassforbothorientinand MassLynx elemental compositioncalculatortoolwithin ppm) errorasillustratedinFigure10.Usingthe mass measurementwasobtainedwithin0mDa(0 obtained waswithin0.2mDa(0.3ppm).ForpeakB, 00 % 0 Passilfora edulis 4 0 .080 00 20 40 60 80 00 20 24.00 22.00 20.00 18.00 16.00 14.00 12.00 10.00 8.00 6.00 .00 21 H 21 ™ O , themostprobableelementalcomposition 11 . Ten majorandminorpeakscontaining A B 21 H 21 Passiflora edulis O 11 21 , theexactmass H 21 O 11 . Fromthe . In CD mined. 100 100 Passiflora edulis. composition forisoorientin(A)foundtobepresentin measurement errorsanddeterminedelemental Figure 9.Exactmassspectrumwith found tobepresent inPassifloraedulis. determined elemental compositionfororientin (B) calculator withmassmeasurement errorsand Figure 11.Illustrationofelemental composition Passiflora edulis. composition fororientin(B)foundtobepresentin measurement errorsanddeterminedelemental Figure 10.Exactmassspectrumwith H % % 0 0 O 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 800 775 750 725 700 675 650 625 600 575 550 525 500 475 450 425 400 375 350 325 300 275 250 225 200 175 150 125 100 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 800 775 750 725 700 675 650 625 600 575 550 525 500 475 450 425 400 375 350 325 300 275 250 225 200 175 150 125 100 H Passiflora edulis Passiflora edulis O H B H O A O OOH OH O OH O H HO O O O OH OH O 3 H 29.0656 OH O O Time H 431.0970 449.1082 449.1084 450.1101 471.0924 4 50.1101 OH OH Error ppm=-0.3 Error mDa=-0.2 Mwt= 449.1084 [M+H]= C21H21O11 A= Isoorientin Error ppm=0 Error mDa=0 Mwt= 449.1084 [M+H]= C21H21O11 B= Orientin m/z m /z

The data produced from the elemental composition calculator is Other potential flavonoids, previously determined to be present shown in Figure 11. Using the resultant exact mass spectrum for within Passiflora species, are isovitexin-2-0-ß-glucopiranoside orientin, the exact mass data was entered into the elemental and , both of which have an elemental composition of composition calculator. An elemental composition is generated C27H30O15. Loss of a glucose molecule would result in a mass and a probability score given, as well as the mass measurement of 433.1135 (C21H21O10) being observed for both molecules, error in mDa and ppm. The elemental composition calculator but this is not the case. However, the fragments observed result was used to generate the probable elemental formula of peaks from successive losses from a glucose moiety of a disaccharide, C and D of Figure 8. i.e. initially m/z 146 and then m/z 163, to produce fragments at m/z 449 and m/z 286 respectively. The same fragmentation Passilfora edulis 595.1658 100 C= Unknown C [M+H]= C27H31O15 pattern observed for flavonoid D also suggests the successive Mwt= 595.1663 Error mDa= -0.5 loss from glucose moieties, but in this case the initial loss being Error ppm= -0.9 from a deoxyhexose group. The proposed structures of flavonoids C and D are illustrated in Figure 15.

Passiflora edulis 449.1088 % 100 D D= Unknown [M+H]= C27H31O14 Mwt= 579.1714 Error mDa= 0 596.1718 449.1089 Error ppm= 0 287.0555 579.1714

674.1401 450.1115 597.1721 334.2025

% 0 m/z 287.0554 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800

Figure 12. Exact mass spectra with mass measurement errors and determined elemental composition for flavonoid (C) found to 450.1140 580.1743

561.1595 be present in Passiflora edulis. 459.1712 601.1526 175.1492 395.2023 411.1960

0 m/z 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 Acquiring full spectra allowed the flavonoid candidates C and D to be distinguished from isoorientin and orientin. The mass Figure 13. Exact mass spectra with mass measurement errors spectra obtained for peaks D and C, along with the generated and determined elemental composition for flavonoid (D) found elemental compositions and mass measurement errors, can be to be present in Passiflora edulis. seen in Figures 12 and 13. The target mass of 449.1084 for orientin and isoorientin was determined within 0.5 mDa for flavonoid C and 0.4 mDa for flavonoid D. In the case of flavonoid C, an elemental composition C27H31O15 was determined to be most probable within -0.5 mDa (0.9 pm). For flavonoid D, C27H31O15 also had a probability score of 1, with mass measurement error of 0 mDa (0 ppm). Figure 14 shows the illustration of elemental composition calculator with mass measurement errors and determined elemental composition for flavonoid (D) found to be present in Passiflora edulis. From the mass spectra acquired for flavonoids C and D, a distinctive fragmentation pattern was observed, possibly caused by thermal degradation or the cone voltage applied. The fragmentation pattern observed was reported by Stoibecki4, for the flavonoid -3-rutinoside. Figure 14. Illustration of elemental composition calculator with mass measurement errors and determined elemental composition for flavonoid (D) found to be present in Passiflora edulis.

ApplicationNOTE Passiflora caerulea Passilfora species. with flavonoidspreviouslyidentifiedtobepresentin Figure 15.ProposedstructuresforflavonoidsCandD m/z 433masschromatogram. Figure 16.Passifloracaeruleaexpandedextracted respectively inFigure17 toFigure21. A, B,C,Dand EwithinFigure16.Theseare illustrated were generated forthefivemajorpeaksidentified as be oneofthetargetflavonoids. Theexactmassspectra minor components.Anyofthe peaks identifiedcould caerulea theycouldbepresent aseithermajoror molecular ionspeciesis433.1135(C glycoside vitexin.Theexactmassoftheprotonated presence ofthe6-Cglycosideisovitexinand8-C chromatogram isillustratedinordertoidentifythe From Figure16them/z433extractedmass If bothvitexinandisovitexinare presentin nominal massofm/z433. major andminorcomponentsbeingidentifiedata clearlydefinable has resultedinatleastfourteen Isolating them/z433extractedmasschromatogram 100 % 0 H H O OH O H H H O O H C O 3 OH O 5 Saponarin H 0 00 50 00 50 00 50 00 50 50.00 45.00 40.00 35.00 30.00 25.00 20.00 15.00 10.00 .00 O O O O H O Flavonoid C OH O H OH O OH OH O O H O O O O A B O C O H O H O H O H H O H HO O O C 3 H H O H O D O O OH Isovitexin-2-0-glucopiranoside O H H OH O O O OH Flavonoid D E 11 O O O H O H H O OH OH 21 Passiflora O O O O O H 10 O H ). OH O H Passiflora caerulea. composition forpeak(A)foundtobepresentin measurement errorsanddeterminedelemental Figure 17.Exactmassspectrumwith elemental compositionofC 433.1135 beingidentified,correspondingtoan mass spectrumgeneratedhasresultedinaof Peak Acouldbeconsideredasvitexinorisovitexin,the flavonoids isovitexinand vitexin. rapid identification ofpeaksBandCtobethe target measurement with fullspectraacquisitionhas allowed candidates tobevitexinorisovitexin. Usingexactmass instrument wouldhaveresulted inmanypotential massrange,onaquadrupole monitoring orashort identity isnotvitexinorisovitexin. Usingsingleion saccharide grouppresenceand loss,indicatingthetrue 563.1765 andm/z595.1714resultingfrom E alsohasionspresentatm/z449.1095, through thelossofanattachedsaccharidegroup.Peak spectrum isadegradationproductofanotherflavonoid 577.1517, indicatingthatthebasepeakofmass Also presentareionsatm/z449.1107and the presenceofionm/z433.1152beingfound. once againillustratesapotentialcandidate,through exact massspectrumpresentedforpeakDinFigure20 the exactmassspectrumhasanerrorof0.5ppm.The 0.2 ppmfromthetargetmass.ShowninFigure19, measurement obtainedwasm/z433.1134,only are potentialcandidates.InFigure18theexactmass C 579.1728, forwhichanelementalcompositionof spectrum itcanbeseenthatactualparentionism/z error fromthetargetmass.Howevermass rhamnoside andV a glucosemoietyofdisaccharidegroup.Vitexin-2-O- 433.1135 indicatesalossofm/z146resultingfrom 100 % 329.5667 0 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 0 2 5 7 800 775 750 725 700 675 650 625 600 575 550 525 500 475 450 425 400 375 350 325 300 275 250 225 200 175 150 Passilflora caerulea 27 A H 31 350.0847 O 14 was produced.Theevidenceofm/z 433.1135 itexin-4-O-rhamnoside (C 370.5977 Time Error ppm=0 Error mDa=0 Mwt=433.1135 C21H21O10 434.1142 21 H 21 5 O 65.1500 579.1728 10 5 80.1750 , whichhaszero 663.1324 27 H Error ppm=2.4 Error mDa=1.4 Mwt=579.1714 A=C27H31O14 7 30 04.1523 O 7 14 15.0969 ) m/z

Passilflora caerulea 433.1134 Passiflora caerulea 433.1139 100 B= C21H21O10 100 B E E=C21H21O10 Mwt=433.1135 Mwt=433.1135 OH Error mDa= -0.1 Error mDa= 0.4 OH HO Error ppm= -0.2 Error ppm= 1.0

O OH OH

HO O E=C27H31O13 Mwt=563.1765 Error mDa= 2.5 OH O Error ppm= 4.5

% % 563.1790

434.1170 434.1179 564.1731 449.1095

595.1714

435.1218 342.0906

0 m/z 0 m/z 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800

Figure 18. Exact mass spectrum with mass measurement errors Figure 21. Exact mass spectrum with mass measurement errors and determined elemental composition for peak (B) found to be and determined elemental composition for peak (E) found to be present in Passiflora caerulea. present in Passiflora caerulea.

Passilflora caerulea Passiflora caerulea 433.1137 100 100 C=21H21O10 C A Mwt=433.1135 B OH Error mDa= 0.2 HO O Error ppm= 0.5 OH

O %

OH O HO OH

OH

0 % 100

% 434.1213

345.5657

303.0500 0 m/z 0 Time 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

Figure 19. Exact mass spectrum with mass measurement errors Figure 22. Passiflora caerulea extract electrospray negative ion and determined elemental composition for peak (C) found to be mode total ion chromatogram and m/z 447 extracted mass present in Passiflora caerulea. chromatogram.

Passiflora caerulea 433.1152 100 A comparative analysis was performed using negative ion mode D D= C21H21O10 Mwt=433.1135 Error mDa= 1.7 electrospray ionization. The negative mode total ion Error ppm= 4.0 chromatogram obtained for the analysis performed of the Passiflora caerulea extract is presented in Figure 22, along with the m/z 447 extracted mass chromatogram showing the presence

% of isoorientin (peak A) and orientin (peak B). The respective mass spectra obtained are illustrated in Figure 23, with the elemental

D= C21H21O11 compositions generated and the mass measurement errors

434.1199 Mwt=449.1084 Error mDa= 2.3 obtained. For isoorientin a mass measurement of 447.0927 Error ppm= 5.2 449.1107 resulted, where the error obtained was 0 mDa (0 ppm). The most

577.1517 0 m/z probable elemental composition for the criteria set was 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800

C21H19O11. For orientin the mass measurement error obtained Figure 20. Exact mass spectrum with mass measurement errors was 0.1 mDa (0 ppm), where C21H19O11 was generated as the and determined elemental composition for peak (D) found to be first elemental composition choice. present in Passiflora caerulea.

ApplicationNOTE Passiflora incarnata 447 extractedmasschromatogram. negative ionmodetotalchromatogram andm/z Figure 24.Passifloraincarnata extractelectrospray 100 100 to bepresentinPassifloracaerulea. composition forisoorientin(A)andorientin(B)found mass measurementerrorsanddeterminedelemental Figure 23.Negativeionexactmassspectrumwith mass chromatogramprofilesof chromatogram arepresented.Them/z449extracted ion chromatogramandm/z447extractedmass and negative ionmodeisshownforthe exampleoftheexactmassdataacquiredin A further of means ofdistinguishingthetwospecies.Incase incar (2.7 ppm)and0.4mDa(0.8ppm). elemental compositionchoicewere1.3mDa forthenumberone measurement errorsdetermined 100 100 % % % % 0 0 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 540 520 500 480 460 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 0 0 Passiflora caerulea Isoorientin Orientin Passiflora incarnata caerulea nata .01.01.02.02.03.03.04.04.050.00 45.00 40.00 35.00 30.00 25.00 20.00 15.00 10.00 5.00 extract showninFigure24,wherethetotal A B are different,andcanbeusedasa , isoorientinandorientinmass Passiflora incarnata Passiflora 447.0929 447.0927 448.0955 448.0920 Error ppm=0 Error mDa=0 Mwt= 447.0927 [M-H]= C21H19O11 A= Isoorientin Error ppm=0.3 Error mDa=0.2 Mwt= 447.0927 [M-H]= C21H19O11 B= Orientin components foundinsomeextractsof per spectra acquisitionandexactmassmeasurementwas allowed forhighlyspecificdatatobeacquired.Full Using oa-TOFLC/MSexactmassmeasurement DISCUSSION orientin, vitexinandisovitexin. of otherflavonoidstobedistinguishedfromisoorientin, ion spectraalsoallowedisobaricdegradationproducts isovitexin. Acquiringfullmassspectradatafragment target flavonoidsisoorientin,orientin,vitexinand acquisition allowedforunequivocalidentificationofthe interest. Thecombinationofexactmassandfullspectra theidentityofflavonoidisomers confirmed derived fromtheexactmassspectrum,thisfurther was calculator themostprobableelementalformula isomer assignment.Usingtheelementalcomposition measurement allowedforfullconfidenceinflavonoid analysis. Theincreasedselectivityofexactmass 1 100 to bepresentinPassifloraincarnata. composition forisoorientin(A)andorientin(B)found mass measurementerrorsanddeterminedelemental Figure 25.Negativeionexactmassspectrumwith 00 % % 0 0 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 540 520 500 480 460 440 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 Passiflora incarnata Isoorientin Orientin formed onmorethaneightymajorandminor formed Time m/z Passiflora 447.0931 447.0940 448.0958 448.0936 Error ppm=2.7 Error mDa=1.3 Mwt= 447.0927 [M-H]= C21H19O11 A= Isoorientin Error ppm=0.8 Error mDa=0.4 Mwt= 447.0927 [M-H]= C21H19O11 B= Orientin in one m /z

CONCLUSIONS ACKNOWLEDGEMENTS

• Real time exact mass centroid data acquisition using positive The authors wish to thank Dr. Ana Maria Soares Pereira and negative ion mode electrospray has been performed. (UNAERP), Prof. Cozimo Pizza (Salerno University) and Dr. Specific parent ion information of the [M+H]+ and [M-H]- ion Laura Meletti (IAC) for providing plant material.

6-C and 8-C -glycoside isomers, as well as other major and The authors wish to thank FAPESP and CNPq for financial minor components in the complex mixtures of the Passiflora support and fellowships. extracts analyzed was obtained in one analysis.

• Acquisition of full exact mass spectral data allowed for REFERENCES isobaric and derivative [1] MA, Y.L.; LI, Q.M.; VANDENHEUVEL, H.; CLAEYS, M. degradation/fragment ions to be distinguished from ions Characterization of flavone and flavonol aglycones by of isovitexin, vitexin, isoorientin and orientin and hence collision-induced dissociation tandem mass spectrometry. correct assignment could be achieved rapidly. Rapid Communications in Mass Spectrometry, v.11, n.12, • Characteristic assignment for 6-C and 8-C flavonoid p.1357-1364, 1997. glycosides isomers (vitexin and isovitexin) (orientin and [2] DOMON, B.; COSTELLO, C.E. A systematic nomenclature isoorientin) has been possible using exact mass for carbohydrate fragmentation in FAB MS/MS spectra of measurement and elemental composition calculation. glycoconjugates. Glycoconjugate Journal, v.5, n .4, • Oa-TOF LC/MS can be used routinely to obtain mass p.397-409, 1988. spectral data within 5 ppm for the characterization of [3] RIRDC Publication No 00/51 RIRDC Project No BRA-3A complex mixtures produced from phytomedicines. [4] STOBIECKI; M. Application of mass spectrometry for the • Isorientin and orientin was identified to be present in identification and structural studies of flavonoid Passiflora caerulea, P.alata, P.edulis and P.incarnata, glycosides. Phytochemistry 54(2000) 237-256 where mass measurement errors of less than 5 ppm were obtained.

• Isovitexin and vitexin were identified to be present in Passiflora caerulea, P.edulis and P.incarnata. For P.alata only isovitexin is present. Mass measurement errors of less than 5 ppm were obtained.

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