Rev. bras. farmacogn. vol.27 número2

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Rapid

identification

and

quantitative

analysis

of

chemical

constituents

of

Gentiana

veitchiorum

by

UHPLC-PDA-QTOF-MS

Shan

Li

a,b

_,

_Chuanxing

_Wan

b

_,

_Lili

_He

c

_,

_Zhigang

_Yan

c

_,

_Kaishun

_Wang

a

_,

_Muhua

_Yuan

a

_,

_Zhifeng

_Zhang

a,∗ a_Institute_of_Ethnic_Medicine,_Southwest_University_for_{Nationalities,}_Chengdu,_China

b_Key_Laboratory_of_Protection_and_Utilization_of_Biological_Resources_in_Tarim_Basin_of_Xinjiang_Production_&_Construction_Group,_Tarim_University,_Alar,_China c_National_Engineering_Laboratory,_Guangxi_Medicinal_Herb_Garden,_Guangxi_Province,_Nanning,_China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5June2016 Accepted23October2016 Availableonline29November2016

Keywords:

Iridoidglycosides FlavonoidC-glycosides Gentiopicroside Isovitexin UHPLC-QTOF-MS

a

b

s

t

r

a

c

t

GentianaveitchiorumHemsl.,Gentianaceae,atraditionalTibetanmedicine,wasusedforthetreatment ofliverjaundicewithdamp-heatpathogen,aswellasforheadacheandchronicpharyngitis.Arapid ultra-performanceliquidchromatography,photodiodearraydetector,quadrupoletime-of-flightmass spectrometrymethodwasdevelopedforthefastandaccurateidentificationandquantificationofthe chemicalconstituentsofG.veitchiorum.Infact,eighteencompoundsweredetectedandidentifiedon thebasisoftheirmassspectra,fragmentcharacteristicsandcomparisonwithpublisheddata. Espe-cially,theMSfragmentationpathwaysofiridoidglycosidesandflavoneC-glycosideswereillustrated. FivecompoundsamongthemwerequantifiedbyUHPLC-PDA,includingswertiamarin,gentiopicroside, sweroside,isoorientin,andisovitexin.Theproposedmethodwasthenvalidatedbasedontheanalyses oflinearity,accuracy,precision,andrecovery.Theoverallrecoveriesforthefiveanalytesrangedfrom 96.54%to100.81%,withRSDfrom1.05%to1.82%.Inaddition,tenbatchesofG.veitchiorumfrom differ-entareaswerealsoanalyzed.Thedevelopedmethodwasrapidandreliableforbothidentificationand quantificationofthechemicalconstituentsofG.veitchiorum,especiallyforsimultaneousqualitativeand quantitativeanalysisofiridoidglycosidesandflavoneC-glycosides.

Introduction

GentianaveitchiorumHemsl.,belongingtothefamilyof Gen-tianaceae,wasknownas“bangjianwenbao”intraditionalTibetan medicine.Itisdistributedataltitudesof2500–4800minQinghai, SichuanandTibetprovincesofChinaandusedforthetreatmentof liverjaundicewithdamp-heatpathogen,aswellasforheadache andchronicpharyngitis.MostChinesepeopledrinkitasaherbal infusion(Liuetal.,2006a,b).

Modernpharmacological studiesshowed thatG. veitchiorum caninhibittheLPSinducedpulmonaryalveolarmacrophages TNF-alphaexpression(Houetal.,2011),theformationofliverfibrosis(Li etal.,2008),andshowedsignificantantibacterialactivityon methi-cillinresistantStaphylococcusaureus(Liuetal.,2011).Meanwhile, theextractofG.veitchiorumdemonstratedsignificantlyanti-RSV effectinvitroandinvivo(Weietal.,2011)andimprovedchronic bronchitis(Gengetal.,2010).Moreover,aformulacontainingthe flowerofG.veitchiorumshowedcertaintherapeuticeffectsagainst

∗ Correspondingauthor.

E-mail:[email protected](Z.Zhang).

pulmonaryinjuriesinducedbybleomycininrats(Liangetal.,2011). OurpreviousstudyhasalsoconfirmedthatG.veitchiorumcould protecttheliveragainstCCl4-induced damageinmice,and this hepatoprotectiveeffectwasduepartlytoitsabilityofscavenging freeradicals(Zhangetal.,2014).

AlthoughpharmacologicaleffectsofG.veitchiorumhavebeen wellinvestigatedinpreviousliteratures,thechemicalanalysesof G.veitchiorumarestill limited.Previous chemicalinvestigations ofG.veitchiorumshowedthatiridoidglycosidesandflavone com-poundswerethemainchemicalconstituents(Zouetal.,2010;Yang etal.,2008).Gentiopicrosidecouldbeoneofthemostimportant chemicalconstituentsofG.veitchiorum.AnHPLCmethodwasalso developedtoquantifygentiopicrosideinG.veitchiorum(Liuetal., 2006a,b).However,theuseofonlyonecompoundasastandard ref-erencefortheevaluationofthechemicalconstituentsincomplex mixtureofherbalmedicinesisnotsufficient(ZhouandHe,2014). Therefore,aUHPLC-QTOF-MSmethodhasbeendevelopedasan efficientandpowerfultoolforsimultaneousqualitativeand quan-titativeanalysisofcomplicatedmixtureofcompoundsinherbal medicines,especiallyforunknowncompounds.

In comparison to previous study, quadrupole time-of-flight massspectrometry(Q-TOF-MS)ismoresensitiveandaccuratevia

http://dx.doi.org/10.1016/j.bjp.2016.10.003

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performingexact mass neutral lossscan togetherwithMS and MS/MSspectra.Moreover,somecompoundscanbeconfirmedby the presences of diagnostic fragments and/or neutral losses in MS/MSspectra.Thecandidate compoundscan befurther iden-tifiedbydetectingexactm/zvaluesfor characteristicfragments and/orneutraleliminations,which arediagnosticforeach class ofnaturalproducts(Zhangetal.,2015;Lietal.,2015).Therefore, inthecurrentstudy,wedevelopedarapidandsensitive UHPLC-PDA-QTOF-MSmethodbasedonexactmassneutrallossscanto simultaneouslyanalyzeknownand unknownconstituents inG. veitchiorumin a shorttime.In total, eighteencompoundswere identifiedortentativelycharacterized.Furthermore,quantitative analysisoffivebioactivecomponentswasperformedby UHPLC-PDA.TenbatchesofG.veitchiorumsamplesfromdifferentcollecting spotswereinvestigatedbytheestablishedmethod.Thepresent studycanprovideimportantinformationforqualitycontrolofG. veitchiorum.Meanwhile,itmightprovidethechemicalevidencefor explainingthetherapeuticeffectsofG.veitchiorum.

Materialandmethods

Plantmaterial

Samplesof Gentianaveitchiorum Hemsl., Gentianaceae,were collectedinSichuanprovince,Qinghaiprovince,Tibetprovinceor purchasedinlocalmarketsattheSichuanprovince.Thematerial wasauthenticatedbyProf.HaoZhang(ProfessorofPharmacognosy, SchoolofPharmacy,SichuanUniversity).Voucherspecimenswere depositedinHerbariumCentre,SouthwestUniversityfor Nation-alities,Sichuan,China.

Chemicalsandreagents

Chemicalreferencestandardsofswertiamarin(No.1402121), gentiopicroside(No.1406017),sweroside(No.1406009), isoori-entin(No.1406014),andisovitexin(No.1406011)werepurchased fromChengduKangbangBiologicalTechnologyCO.,LTD(Chengdu, China).Thepurityofeachchemicalwasabove98%,asconfirmed byHPLC.MethanolwasHPLCgradefromMerck(Darmstadt, Ger-many). Water was obtained from a Milli-Q water purification system(Millipore,Bedford,MA,USA).Otherreagentsand chem-icalswereofanalyticalgrade.Allsolutionswerepassedthrough a0.22␮mPTFEmembrane(CherishTechnologyCO.,LTD,Beijing, China)beforeinjectingintotheUHPLCsystem.

Preparationofstandardsolutions

Standardsofswertiamarin,gentiopicroside,sweroside, isoori-entin,andisovitexinweredissolvedinmethanoltoproducefive stocksolutions(about1mg/ml),whichwerestoredat4◦_C._Working solutionsofstandardswerepreparedbydilutingthestocksolutions withmethanolintodifferentconcentrations.

Preparationofsamplesolutions

G.veitchiorumwascollectedandthendried.Thesampleswere cut into small sections and ground to powder in a mill. The powder(0.5g)wasextractedwithmethanol(2×10ml)by son-ication (30min, at room temperature), the solutions combined andadjustedto25mlvolume.Thesamplesolutionswerefiltered througha0.22␮mPTFEmembranefilter.Thefiltratewasstoredat 4◦_C_in_a_refrigerator_before_being_used_for_UHPLC_analysis.

MethodvalidationofUHPLC-PDA

FollowingtheanalyticalprocedureguidelineofICH2005,the developedUHPLC-PDAmethodwasvalidatedbasedonthe linear-ity,precision,repeatability,andrecovery.

(1)Limitsofquantificationanddetection

The standards stock solutions were diluted toa series of appropriateconcentrationswithmethanol.Thediluted solu-tionswereanalyzedbyUHPLC.Thelimitofdetection(LOD)and limitofquantification(LOQ)weredeterminedbyinjectingthe dilutedsolutionwithknownconcentrationsofthestandards. LODandLOQweredefinedasthesignal-to-noiseratios(S/N) equalto3and10,respectively.

(2)Linearity

The stock solutions of the five standards weremixed for construction of calibrationcurves. Sixlevels ofthe solution concentrationweredeterminedinduplicate,and calibration curveswerethenestablishedbyplottingthepeakareaversus theconcentrationofeachanalyte.Theregressionequationwas calculatedasfollows:Y=aX+b,whereYisthepeakareaandX istheconcentrationofthestandardsolutions.

(3)Precision,repeatabilityandrecovery

Theintra-andinter-dayprecisionswereinvestigatedby ana-lyzing a mixedstandard solutionin fivereplicates duringa singledayandbyduplicatingtheexperimentsonthree con-secutive days.G.veitchiorumwasanalyzedin fivereplicates withtheproposedmethodtoconfirmtherepeatabilityofthe developedapproach.Therelativestandarddeviation(RSD)was calculatedasavariationofprecisionandrepeatability.A recov-eryexperimentwascarriedouttofurtherevaluatetheaccuracy ofthemethod.Threedifferentquantities(low,medium,and high)ofthestandardswerespikedintoaknownamountof G.veitchiorumsample(0.25g).Then,theresultantsamplewas extractedandanalyzedwiththeestablishedmethodand trip-licateexperimentswereperformedateachlevel.Therecovery percentagesforthefivecompoundswerecalculatedbasedon thefollowingequation:

Recovery (%)= detectedamount-original_spiked_amount amount×100.

UHPLC-PDA-QTOF-MSanalysis

UHPLC-PDA-QTOF-MSanalysiswasperformedontheWaters AcquityUHPLCI-Classsystem(Waters,USA).Samplewasseparated inanAcquityHSSC18column(100mm×2.1mm,1.8␮m)witha C18pre-column(Waters,USA).Thecolumn wassetatthe tem-peratureof35◦_C._The_mobile_phase_was_a_mixture_of_0.1%_formic acid–water(A)andmethanol(B),withanoptimizedlineargradient elutionasfollows:0–15min,20–50%B.Theinjectionvolumewas 1.0␮l.Theflowratewas0.20mlmin−1_._Detector_wavelength_was setat254nm.

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Table1

Linearregression,precision,repeatability,andrecoverydataoffiveanalytesfromGentianaveitchiorum.

Compounds Regressionequations r Linearrange Precision(RSD) Repeatability Recovery LOD LOQ

(␮gml−1₎ _Intra-day _Inter-day _(RSD) _Average _(RSD) ₍_␮_g_ml−1₎ ₍_␮_g_ml−1₎

Swertiamarin Y=8.1711×10−6_X₋_315,852.9 _0.9998 _0.07–2.30 _2.03% _2.58% _2.45% _100.04% _1.82% _0.51 _1.26

Gentiopicroside Y=3.7819×10−6_X₋_11,742.0 _0.9997 _0.15–2.36 _1.34% _2.30% _1.67% _100.81% _1.91% _0.46 _1.03

Sweroside Y=7.6114×10−6_X₋_10,172.2 _0.9995 _0.06–2.00 _1.56% _2.71% _1.38% _98.78% _1.33% _0.53 _1.65

Isoorientin Y=9.5069×10−6_X₋_102,206.5 _0.9999 _0.09–2.01 _1.18% _2.01% _2.13% _98.12% _1.12% _0.33 _1.43

Isovitexin Y=6.4399×10−6_X₋_11,628.2 _0.9999 _0.11–2.12 _1.31% _2.41% _1.28% _99.11% _1.05% _0.56 _1.01

Resultsanddiscussion

MethodvalidationofUHPLC-PDA

Toachievethebestresolutionforeachdetectedpeakswithin

shortertime,theorganicsolvent,columntype,mobilephase,flow

rateofmobilephase,andcolumntemperaturewereoptimized.The

developedmethodforthequantificationofanalyteswasvalidated

bydeterminingthelinearity,limitofdetection(LOD),limitof

quan-tification(LOQ),intra-dayandinter-dayprecisions,repeatability

and recovery. Swertiamarin, gentiopicroside, sweroside,

isoori-entinandisovitexinshowedgoodlinearitywithhighcorrelation

coefficient(r>0.9999)withinthetestedrange.TheLOD(S/N=3)

andLOQ(S/N=10)forthefivestandardanalyteswereintherange

of0.33–0.56ng/mland1.01–1.65ng/ml,respectively.

Precision and repeatability of theUHPLC-PDA methodwere

alsovalidatedforthefiveanalytes.Relativestandard deviations

(RSD) of intra-day, inter-day precision, and repeatability were

1.18–2.03%,2.01–2.71%,and1.28–2.45%,respectively, indicating

highprecisionandgoodrepeatability.Thefiveanalyteswere

sta-bleinpreparedsamplesolutionwhenplacedintheautosamplerat

4◦_C_for₂₄_h,_with_the_RSD_of_{1.18–2.13%.}_Five_replicate_tests_were

performed.Thedevelopedmethodwasalsoaccuratewith

recov-eriesof98.12–100.81%,andwiththeRSDof1.05–1.91%.Theabove

datashowedthatthedevelopedmethodwassensitiveand

accu-rateforquantitativedeterminationoftheseanalytes.Theresults

aresummarizedinTable1.

Quantitativeanalysis

ThedevelopedUHPLC-PDAanalyticalmethodwassubsequently applied to the simultaneous determination of G. veitchiorum fromdifferentregionsinChina.Therepresentativechromatogram was shown in Fig. 1(A,C). The five analytes (swertiamarin,

1.5e-1

A

B

C

(1) PDA ch1 [email protected] Range: 2e-1

Time

10

5

10

9

7

5 8

6 12

11 13

15

14

17 16

18 4

10

8 11 17

18 3

2 1

8

6 11

12 13

14 15

17

18

1.25e-1

1.0e-1

AU 7.5e-2

5.0e-2

2.5e-2

0.0

98

–2

–0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

%

–0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

1.5e-1

1.25e-1

1.0e-1

AU 7.5e-2

5.0e-2

2.5e-2

0.0

–0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 1: TOF MS

ES-BPI 1.57e6

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Table2

Quantificationofswertiamarin,gentiopicroside,sweroside,isoorientin,andisovitexinfromdifferentspotsamplesofGentianaveitchiorum.

Samples Swertiamarin Gentiopicroside Sweroside Isoorientin Isovitexin

(mgg−1₎ _(mg_g−1₎ _(mg_g−1₎ _(mg_g−1₎ _(mg_g−1₎

S2015001fromHongyuan,Sichuan 5.49±0.02 2.69±0.02 5.82±0.13 6.02±0.09 3.69±0.02

S2015002fromKangding,Sichuan 5.06±0.05 3.01±0.03 5.56±0.22 5.81±0.14 3.01±0.08

S2015003fromLuhuo,Sichuan 2.62±0.02 2.47±0.02 4.92±0.15 5.92±0.05 2.66±0.05

S2015004fromRuoergai,Sichuan 4.89±0.04 2.46±0.04 5.52±0.21 4.82±0.12 2.46±0.07

S2015005fromHainanzhou,Qinghai 2.01±0.10 2.23±0.02 2.44±0.08 3.74±0.08 3.21±0.04

S2015006fromLinzhi,Tibet 3.07±0.12 3.01±0.06 3.51±0.14 2.91±0.14 3.00±0.08

S2015007fromlocalmarketofSichuan 2.13±0.09 2.54±0.06 4.15±0.21 3.15±0.03 2.50±0.11

S2015008fromlocalmarketofSichuan 2.98±0.05 2.21±0.09 3.67±0.16 3.67±0.18 2.44±0.12 S2015009fromlocalmarketofSichuan 2.24±0.03 2.78±0.09 3.24±0.16 2.34±0.16 2.61±0.03 S2015010fromlocalmarketofSichuan 3.65±0.11 2.12±0.02 4.12±0.43 4.21±0.07 2.66±0.05

gentiopicroside,sweroside,isoorientin,andisovitexin)were

repre-sentativecompoundsofiridousandflavoneglycosidesinthefamily

ofGentianaceaeandhadbeenreportedtoexhibitsignificantly

cyto-protectiveeffects(Oztürketal.,2006).Therefore,thedeveloped

UHPLC-PDAmethodcouldbeusedforcomprehensiveevaluation ofthequalityofG.veitchiorum.Theresultsofdetermining stan-dardsanalytesofG.veitchiorumfromdifferentcollectingspotswere showninTable2.Itwasfoundthatthefiveanalytescanbedetected inallofthesamples.Thetotalcontentsofthefiveanalytesin sam-plesfromHongyuanandKangdingcountiesofSichuanprovince werehigherthanthevaluesfromotherplaces.Thecontentsofthe fiveanalytesofsamplesfromQinghaiwerethelowest.The differ-entiationmaybeduetodifferencesinclimate,soil,andecological environment.

IncontrastwitharecentstudythatreportedtheuseofHPLC coupledwithUV toestablishchromatographic fingerprintsand determiningthecontentofonecompoundinG.Veitchiorum(Wang, 2010),thepresentworkidentifiedeighteencompoundswithina shortperiod of 15min,particularly, thesimultaneous quantita-tiveanalysisofthefivemainconstituentswhichweredifficultto isolateusingconventionalmethods.Thisresultindicatedthatthe developedmethodwasmoresensitiveandtime-savingthanthe previouslyreportedprocedure.

IdentificationofthedetectedcomponentsofGentiana veitchiorum

TheethanolextractsofG.veitchiorumwereanalyzedbyUHPLC gradient elutionwith QTOF-MS detection for thefirst time. To achievethehighestsensitivityforthechemicalconstituents,the ionizationofcompoundsofG.veitchiorumwasperformedinboth negativeandpositiveionmodes.Theresultsshowedthatnegative modewasmoresensitiveforthedetectionofthesecompounds. TypicalMSbasicpeakion(BPI)chromatogramtrace,with num-beredpeaksofG.veitchiorum,wasillustratedinFig.1(B).

Intotal,eighteencompoundswerewelldetectedinthe devel-opedmethodandtentativelyorunambiguouslycharacterizedfrom G. veitchiorum. Formic acid was added in the mobile phase in ordertoimprovechromatographicpeakresolutionandgenerate adducts[M−H+HCOOH]−,which werehelpfulforthe confirma-tionofdeprotonated[M−H]−_,_protonated_ions_[M+H]+_or_sodium adducts[M+Na]+_.

Compounds1and2at1.31minand1.42mindisplayedasimilar deprotonatedion[M−H]−_at_m/z_301.0914_and_301.0918_with_the samemolecularformulaC13H18O8.Thefragmentinformationof

1atm/z139.0391[M−H−162]−_and_124.0155_[M₋_H₋₁₆₂₋_15]− couldbeexplained bytheloss ofone glucosyltogether witha methylgroupswhilecompound2wasobservedthelossofthe agly-contoformglucosylionsatm/z179.0555[Glu−H]−_and_161.0443 [Glu−H−H2O]−.Compounds1and2shouldbeassignedasthe iso-mersviatheirhigh-resolutionmassvalues.TheClogPvaluesofthe

twocompoundswerecalculatedusingChemDrawsoftware. Com-pound1hassmallerClogPvaluewhileCompound2hashigher value.Theywere−0.8251and−0.7251,respectively.TheClogP valueofonecompound,whichwasthelogarithmofitspartition coefficientbetweenn-octanolandwaterlog(Coctanol/Cwater),wasa well-establishedmeasureofacompoundhydrophilicity.Therefore, compound1wasfirstlyeluted.Compounds1and2weretentatively identifiedasisotachiosideandtachioside(Xiaoetal.,2010), respec-tively.Thetwocompoundswereseparatedandidentifiedforthe firsttimeinG.veitchiorum.

Compound3wasidentifiedasanorganicacid,whichshowed adeprotonatedion[M−H]−_at_m/z_315.0707_with_the_molecular formulaC13H16O9.Thefragmentionatm/z153.0190suggestingthe lossofaglucoseresidue[M−H−162]−_._The_fragment_information atm/z109.0284[M−H−162−CO2]−couldbeexplainedbytheloss ofcarbondioxide.Compound3wastentativelyidentifiedas 5-(␤-d-glucopyranosyl)-2-hydroxybenzoicacid(Yeonetal.,2013).

Mass spectrum of compound 4 showed thedeprotoned ion [M−H]−atm/z537.1819withthemolecularformulaC23H34O15, Thefragmentinformationatm/z375.1302[M−H−162]−_and_m/z 213.0771[M−H−162−162]−_could_be_explained_by_the_consecutive eliminationoftwohexoses.Thestructureofthiscompoundwas tentativelyidentifiedas6′_-O-␤-d-glucosyl-loganicacidandithas beenpreviouslyreportedintheGentianaceaefamily(Suryawanshi etal.,2006).

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Compound6at2.97minshowedanadduct atm/z563.1611 [M−H+HCOOH]− _which _confirmed _the _molecular _formula C22H30O14 in the negative ionization mode. Sodium adduct at m/z 541.1531 [M+Na]+ _was _observed _in _positive _ionization mode. Further CID–MS2 _scan _displayed _fragment _ions _at _m/z 341.1089 [2Glu−H]− and m/z 179.0557 [Glu−H]−, indicating two glucosyl groups. The further fragments at m/z 161.0445, 149.0594, 119.0338, 112.9845 were produced from the gluco-syl ion. Therefore, compound 6 was tentatively identified as 6′_-O-␤-d-glucosyl-gentiopicroside(Kakudaetal.,2001).

Compound7,thedeprotonatedionpeakwasobservedatm/z 293.1231[M−H]−_with_the_molecular_formula_C₁₂_H₂₂_O₈_and frag-mentionsatm/z248.9602couldbeexplainedbythelossofcarbon dioxide[M−H−CO2]−.Thestructureofthiscompoundwas tenta-tivelyidentifiedas2-O-glucosyl-hexanoicacid.

Compounds9and15at3.74minand5.55mindisplayeda sim-ilardeprotonatedion peak atm/z755.2043 and 755.2044with thesamemolecularformulaC33H40O20.Thefragmentation infor-mation at m/z 431.0984 [M−H−2Glc]− could be explained by thelossoftwo O-glucosylgroups, and thestrongpeaksatm/z 341.0653[M−H−2Glc−90]−_and_m/z_311.0557_[M₋_H₋_2Glc₋_120]− indicatedanotherC-glucosylgroup.Moreover,theionsof9and

15atm/z413.0866and413.0872[M−H−2Glu−H2O]−confirmed theexistenceoftheC-glucosylgroupandlocateditspositionat C-6of theflavone skeleton (Cuyckens and Claeys, 2004). Com-pounds9and15maybetheisomerswiththreeglucosylgroups. TheClogPvaluesofthetwocompoundswerecalculated. Com-pound9 has thesmallerClog Pvalue while compound 15 has thehigherClogPvalue.Theywere-2.93and-2.87,respectively. Therefore,compounds9and15weretentativelyidentifiedas iso-vitexin4′_{-O-diglucoside}_and_isovitexin_{7-O-diglucoside}_(Xu_et_al., 2009).

Compound 12 at 4.66min presented a deprotonated ion peak at m/z 593.1504 with a molecular formula C27H30O15. The fragmentation information at m/z 503.1197 [M−H−90]−, 473.1085[M−H−120]−,341.0661[M−H−90−162]−,and311.0551 [M−H−120−162]−_indicated_one_C-glucosyl_group_together_with anO-glucosylgroup(CuyckensandClaeys,2004).Fragmentalions atm/z282.0522and283.0571weresimilartocompound18, sug-gesting theaglycon to bethesame as that of isovitexin. Thus, thestructureofthiscompoundwastentativelyconfirmedas isos-aponarin(Bergeronetal.,1997).

Compounds13and16at4.88minand6.36mindisplayedtheir deprotonatedionpeaksatm/z609.1457 and609.1446withthe same molecular formula C27H30O16. The fragment information atm/z447.0926[M−H−162]− _and _285.0393_[M₋_H₋₁₆₂₋_162]− couldbeexplainedbythelossoftwoconsecutivehexoseunits. Twostrongpeaksatm/z357.0602[M−H−162−90]−,and327.0499 [M−H−162−120]− indicated that one of the hexoses was C-glucosyl,the otheris O-glucosylgroup. Compounds13 and 16

mustbeisomers.Compound13hasthesmallerClogPvaluewhile compound16hasthehigherClogPvalue.Theywere−1.98and −1.63,respectively.Therefore,compounds13and16were tenta-tivelyidentifiedasisoorientin4′_-O-glycoside_{(Schaufelberger}_and Hostettmann,1987)andlutonarin(Liuetal.,2005),respectively.

Thetwocompoundswereseparatedandidentifiedforthefirsttime inG.veitchiorum.

Compound 14 displayed a molecular ion at m/z 771.1984 [M−H]− _with _a _molecular _formula _C

33H40O21. The deproto-natedion [M−H]−_showed_the_fragmental_ions_of_m/z_447.0927 [M−H−2Glc]−,m/z357.0605[M−H−2Glc−90]−,andm/z327.0499 [M−H−2Glc−120]−.Moreover,theaglyconionofcompound 14

was very similar withthat observed for compound 17. There-fore, compound 14 was tentatively identified as isoorientin 4′_-diglucoside_(Xu_et_al.,_2009).

Thedeprotonatedionpeakofcompound17wasobservedatm/z 447.0925[M−H]−_with_the_molecular_formula_C₂₁_H₂₀_O₁₁_and frag-mentionsatm/z357.0601[M−H−90]−,327.0503[M−H−120]− and 285.0393 [M−H−162]−_, _which _could _be_explained _by par-tially or fully losing of a C-glucosyl group. In addition, RDA cleavageionsofflavonoidswerealsoobservedatm/z161.0244 and 117.0335. The structure of this compound was tenta-tively confirmed as isoorientin, and it has been previously reported in Gentianaceae (Wang et al., 2008; Sasaki et al., 2015).

Compound 18 displayed a deprotonated ion peak at m/z 431.0976[M−H]−_with_a_molecular_formula_C₂₁_H₂₀_O₁₀_. Further-more,CID–MS2_scan_showed_that_they_produced_fragment_ions_at m/z311.0554[M−H−90]−_and_283.0601_[M₋_H₋_120]−_showing C-glucosylgroup,togetherwithRDAcleavageionsofflavonoidsat m/z161.0244and117.0335,whichagreedwithpreviouslyreported dataforisovitexin,whichhasbeenreportedintheGentianaceae family(Huangetal.,2015).

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Table3

CharacterizationofchemicalconstituentsinG.veitchiorumbyUHPLC-QTOF-MS.

PeakNo. Retention (min)

Molecular formula

[M−H]−_(error_(ppm)) _MS/MS_fragments_ions _[M+Na]+ _Identified_compounds

1 1.31 C13H18O8 301.0914(−3.0) 139.0391,124.0155 325.0894 Isotachioside

2 1.42 C13H18O8 301.0918(−1.7) 179.0555,161.0443,121.0283, 119.0338

325.0894 Tachioside

3 1.57 C13H16O9 315.0707(−2.9) 153.0190,109.0284 5-(ˇ-d

-glucopyranosyl)-2-hydroxybenzoic acid

4 2.40 C23H34O15 537.1819(0.0) 375.1302,213.0771,169.0865,

129.9758,113.0236

6′_-_O_-_ˇ_-d-glucosyl-loganicacid

5 2.63 C16H24O10 375.1283(−2.1) 213.0756,169.0860,151.0754, 113.0232

399.1270 Loganicacid

6 2.97 C22H30O14 563.1611(−0.7) 517.1556,341.1089,221.0656, 179.0557,161.0445,149.0594, 119.0338,112.9845

541.1531 6′_-_O_-_ˇ_-d-glucosyl-gentiopicroside

[M−H+HCOOH]−

7 3.11 C12H22O8 293.1231(−1.7) 248.9602,229.0387,131.0701, 113.0231

2-O-glucosyl-hexanoicacid

8 3.26 C16H22O10 419.1182(−1.9) 409.0893,179.0551,161.0446, 141.0180,119.0336,112.9843

Swertiamarin

[M−H+HCOOH]−

9 3.74 C33H40O20 755.2043(−1.3) 431.0984,413.0866,341.0653,

311.0557,146.9635,112.9853

Isovitexin4′_-_O_-diglucoside

10 3.89 C16H20O9 401.1081(−0.7) 391.0801,355.0004,179.0558 379.1001 Gentiopicroside [M−H+HCOOH]−

11 4.48 C16H22O9 403.1234(−1.5) 393.0944,357.1202,195.0662, 125.0232

409.1588 Sweroside

[M−H+HCOOH]−

12 4.66 C27H30O15 593.1504(−0.3) 503.1197,473.1085,341.0656,

311.0551,283.0571,282.0522

617.1483 Isosaponarin

13 4.88 C27H30O16 609.1457(0.3) 489.1037,447.0926,357.0605,

327.0499,298.0467,285.0393

633.1427 Isoorientin4′_-_O_-glycoside

14 5.43 C33H40O21 771.1984(0.9) 447.0927,429.0822,357.0605,

327.0499,285.0394,112.9850

485.1429 Isoorientin4′_-diglucoside

15 5.55 C33H40O20 755.2044(1.2) 575.1420,431.0979,413.0872,

341.0658,311.0553,282.0522, 161.0239,112.9847

Isovitexin7-O-diglucoside

16 6.36 C27H30O16 609.1446(−1.6) 519.0013,489.1036,447.0926, 357.0602,327.0496,284.0318

633.1429 Lutonarin

17 8.22 C21H20O11 447.0925(−0.4) 357.0601,327.0494,298.0469, 285.0393,163.0030,133.0282

471.0900 Isoorientin

18 10.47 C21H20O10 431.0976(−0.5) 311.0548,283.0601,161.0244,

117.0335

455.0945 Isovitexin

Conclusions

Thepresentworkisthefirstreportofanaccurate,rapid,and

reliableanalyticalmethodforthesimultaneousquantitationand

identificationofchemicalconstituentsinG.veitchiorumby

UHPLC-PDA-QTOF-MS.Eighteencompoundsweresuccessfullyseparated

byUHPLCandidentifiedortentativelycharacterizedbyperforming

exactmassneutrallossscantogetherwithMSandMS/MS

spec-traofQTOF-MS,includingsixiridoidglycosides,eightflavonoids,

threephenolicglycosides,andoneglucosylatedorganicacid.Three

iridoidglycosidesandtwoflavonoidsweresimultaneously

deter-minedandquantifiedinallsamplesunderonerun.Therefore,the

developedmethodwasausefulandreliablequalitycontrolmethod

forarapididentificationandquantificationofthechemical

con-stituentsofG.veitchiorum.

Authorscontributions

LSperformedthelaboratoryworkanddraftedthepaper.WCX

contributedinMSdataanalysis;HLLandYZGwereresponsible

forHPLCdataanalysis;WKSandYMHcollectedalloftheplant

samples;ZZFdesignedthestudyandsupervisedtheoverallproject

work.Authorshavereadthefinalmanuscriptand approvedthe

submission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Thisstudywassupportedbythefunds ofBasicResearchfor

Application of Sichuan Province (2014JY0233), Natural Science

FoundationofGuangxi(2015GXNSFBA139118)andKeyR&D

Pro-gramofGuangxi(GuiBranchAB16380346).

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