Linear Quantitative Profiling Method Fast Monitors Alkaloids of Sophora Flavescens That Was

15 Pages·2016·1.62 MB·English

RESEARCHARTICLE
Linear Quantitative Profiling Method Fast
Monitors Alkaloids of Sophora Flavescens
That Was Verified by Tri-Marker Analyses
ZhifeiHou1,2,GuoxiangSun1*,YongGuo3
1 SchoolofPharmacy,ShenyangPharmaceuticalUniversity,Shenyang,China,2 Departmentof
PharmaceuticalEngineering,HebeiChemicalandPharmaceuticalCollege,Shijiazhuang,China,3 Schoolof
Pharmacy,FairleighDickinsonUniversity,FlorhamPark,NewJersey,UnitedStatesofAmerica
*[email protected]
Abstract
ThepresentstudydemonstratedtheuseoftheLinearQuantitativeProfilingMethod
a11111 (LQPM)toevaluatethequalityofAlkaloidsofSophoraflavescens(ASF)basedonchro-
matographicfingerprintsinanaccurate,economicalandfastway.Bothlinearqualitative
andquantitativesimilaritieswerecalculatedinordertomonitortheconsistencyofthesam-
ples.Theresultsindicatethatthelinearqualitativesimilarity(LQLS)isnotsufficientlydis-
criminatingduetothepredominantpresenceofthreealkaloidcompounds(matrine,
sophoridineandoxymatrine)inthetestsamples;however,thelinearquantitativesimilarity
OPENACCESS (LQTS)wasshowntobeabletoobviouslyidentifythesamplesbasedonthedifferencein
thequantitativecontentofallthechemicalcomponents.Inaddition,thefingerprintanalysis
Citation:HouZ,SunG,GuoY(2016)Linear
wasalsosupportedbythequantitativeanalysisofthreemarkercompounds.TheLQTS
QuantitativeProfilingMethodFastMonitorsAlkaloids
ofSophoraFlavescensThatWasVerifiedbyTri- wasfoundtobehighlycorrelatedtothecontentsofthemarkercompounds,indicatingthat
MarkerAnalyses.PLoSONE11(8):e0161146. quantitativeanalysisofthemarkercompoundsmaybesubstitutedwiththeLQPMbasedon
doi:10.1371/journal.pone.0161146
thechromatographicfingerprintsforthepurposeofquantifyingallchemicalsofacomplex
Editor:EtsuroIto,WasedaUniversity,JAPAN samplesystem.Furthermore,oncereferencefingerprint(RFP)developedfromastandard
Received:March28,2016 preparationinanimmediatedetectionwayandthecompositionsimilaritiescalculatedout,
LQPMcouldemploytheclassicalmathematicalmodeltoeffectivelyquantifythemultiple
Accepted:August1,2016
componentsofASFsampleswithoutanychemicalstandard.
Published:August16,2016
Copyright:©2016Houetal.Thisisanopenaccess
articledistributedunderthetermsoftheCreative
CommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
Introduction
medium,providedtheoriginalauthorandsourceare
credited. ThedriedrootofSophoraflavescensAit.,knownasKuShen(SophoraeFlavescentisRadix)in
DataAvailabilityStatement:Allrelevantdataare China,iswidelyusedinTraditionalChineseMedicine(TCM)foritseffectofclearingheatand
withinthepaperanditsSupportingInformationfiles. dampness,killingparasites,andinducingdiuresis[1].Modernresearchhasalsoshownvarious
Funding:Theauthorshavenosupportorfundingto pharmacologicaleffectsofAlkaloidsofSophoraflavescens(ASF),suchasantimicrobial,anti-
report. inflammatory,anti-allergic,anti-tumor,anti-arrhythmia,anti-hepatitis,andregulationofthe
immunesystem[2–17].AmongmanychemicalcomponentsfromtheASF,quinolizidinealka-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. loidssuchasmatrine(MT),sophoridine(SPR)andoxymatrine(OMT)havebeen
PLOSONE|DOI:10.1371/journal.pone.0161146 August16,2016 1/15
LinearQuantitativeProfilingMethodMonitorsAlkaloidsofSophoraFlavescens
demonstratedtobeimportantactivecompounds[18–29].ThequalityofASFasherbalmedi-
cineistypicallyevaluatedandcontrolledbythetotalalkaloidcontent.Infact,theNational
DrugStandardforASFinChinaisbasedondeterminingthetotalalkaloidcontentusinga
titrationmethod[30].Inpractice,thequalityofASFistypicallyevaluatedbasedonthequanti-
tativecontentofthemarkercompounds(e.g.,MT,OMTandSPR)determinedbyhigh-perfor-
manceliquidchromatography(HPLC)orcapillaryelectrophoresis(CE)method[31,32].But
quantitativedeterminationofmultiplemarkercompoundsrequiressignificantamountof
effortsandresources.Inadditiontoquantitativeanalysisofthemarkercompounds,chro-
matographicfingerprinthasbeenwidelyadoptedforqualityassessmentofherbalmedicinesby
regulatoryagencies,suchasUSFoodandDrugAdministration(FDA),EuropeanMedicines
Agency(EMA),andChinaFoodandDrugAdministration(CFDA)[33].However,thechro-
matographicfingerprintsarecommonlyanalyzedforqualitativesimilarityamongthetestsam-
ples.Thequantitativecontentsofthechemicalcomponentsinherbalmedicinesarenot
analyzedtocomparethesimilarityofthesamples.Forexample,Zhangetalusedaqualitative
hierarchicalclusteringmethodtoanalyzethechromatographicfingerprintsofthealkaloids
fromcommercialSophoraeFlavescentisRadix,andalsodeterminedthecontentofmultiple
markercompoundsforthepurposeofqualityassessment[34].Theyconfirmedtheusefulness
ofchromatographicfingerprintinginqualityevaluationofherbalmedicines,butalsoacknowl-
edgedthatdirectquantitationofmultiplemarkercompoundswasnotalwaysfeasibledueto
theavailabilityofthestandardsforthemarkercompounds.
Thechromatographicfingerprintsoftheherbalmedicinesamplesaretypicallyusediniden-
tificationandauthenticity[35–36],whichonlyindicatesthequalitativesimilarityinthepres-
enceanddistributionofthechemicalcomponents.Thedifferenceinthequantityofthe
chemicalcomponentsisnotaddressedbyqualitativesimilarityanalysis.Butthisisnotthecase
currently.Quantitativesimilarityanalysishasbeendevelopedasameasuretodetectthediffer-
enceinthequantitativecontentsoftheherbalsamplesandhasbeensuccessfullyappliedto
qualityevaluationofsometraditionalChinesemedicines[37–39].Inthisstudy,theLinear
QuantitativeProfilingMethod(LQPM)wasfirstusedtoevaluatebothlinearqualitativeand
quantitativesimilaritiesofthechromatographicfingerprintsoftheASFsamples.Threealka-
loidmarkercompounds(MT,SPRandOMT)werealsoquantitatedusingavalidatedHPLC
method,andtherelationshipbetweenthelinearquantitativesimilarity(LQTS)andthecontent
ofthemarkercompoundswasalsoinvestigated.LQPMisalowconsumedandhighlyeffective
waycomparedwithanymulti-markerquantitativelydeterminationmethod.Asacomplex
multiplecomponentsystem,ASFsamplescanbefirstlyidentifiedandsecondlyquantifiedby
LQPMfromanoverallmode.QualityconsistencyofASFsampleshasbeensuccessfullymoni-
toredandeffectivelyprovedbyourresearchresults.
TheLinearQuantitativeProfilingMethod
X~¼ðx1;x2;(cid:2)(cid:2)(cid:2);xnÞandY~¼ðy1;y2;(cid:2)(cid:2)(cid:2);ynÞserveasthesampleandreferencevectorwherexi
andyiaretheithpeakareainthesamplefingerprint(SFP)andreferencefingerprint(RFP),
respectively.ThecorrelationcoefficientrforlinearequationX~¼aþbY~canbecalculated
accordingtoEq1andrepresentsalinearqualitativesimilarity(LQLS),whichdescribesthedis-
tributioncharacteristicsofallchemicalfingerprintsintheherbalmedicines.Theslopeb,calcu-
latedaccordingtoEq2,canbeusedtoquantitativelycompareX~andY~aftercorrectionofthe
apparentweightofSFP(mj)andRFP(mR).Theparametermjistheweightofthejthsample
andtheparametermRistheaverageweightof27batchesofsamples.Therefore,theslopebis
calledaslinearquantitativesimilarity(LQTS)andcanbeusedtomeasurequantitativesimilar-
ityinthetotalcontentofallfingerprintcomponentsbetweenSFPandRFP.Infact,bisvery
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LinearQuantitativeProfilingMethodMonitorsAlkaloidsofSophoraFlavescens
Table1. ThequalitygradeassignedbyLQPMandtheacceptancecriteria.
Qualitygrade 1 2 3 4 5 6 7 8
best better good fine moderate common inferiors defective
r(cid:6) 0.95 0.90 0.85 0.80 0.70 0.60 0.50 <0.50
b2 95*105 90*110 85*115 80*120 70*130 60*140 50*150 0*1
α(cid:7) 0.05 0.10 0.15 0.20 0.30 0.40 0.50 >0.50
doi:10.1371/journal.pone.0161146.t001
closetotheapparentcontentsimilarityR%(seeninEq3),andtheerrore(beequalto
¼R(cid:3)b¼að(cid:2)yÞ(cid:3)1(cid:4)100%)isaboutlessthan3%.Thetermαisastatisticalerrorcalculated
accordingtoEq4andreflectstheaccuracyofthelinearmodel.Thelinearqualitativesimilarity
(r),linearquantitativesimilarity(b)anderrorterm(α)arecombinedintheLinearQuantita-
tiveProfilingMethod(LQPM)toevaluatethequalityoftheherbalmedicines(8gradeslisted
inTable1).
Infact,risalwayslessthanunity,howeverbcanbeinrangeof0—1,thusthereisnearly
anorthogonalitycorrelationbetweenrandb,whichindicatesthattheLQTSassessismore
importantthanLQLS.Inaddition,asresponsesofdifferentdetectorsaredissimilartovarious
fingerprintcomponents,weightcorrectionfactorshouldbetakenintoconsideration.However,
theweightcorrectionfactorcanbeignoredforthehomologuefingerprintslyinginprofiles.
ThecompositionsimilaritiesofLQLSandLQTScanbeusedtoanalysethecontributionof
somefingerprintpeaks.Theycanbecalculatedoutatoncewhenonefingerprintpeakareawas
inputintothenumeratorinEqs1and2,respectively,whilethedenominatorwereunchanged.
ThereforebothLQLSandLQTSrobustlyconstructthetwodimentionalorthogonalsimilarities
forreasonablyevaluatingfingerpringprofilingofTCMandherbalmedicines.
P
n ðx (cid:3)~xÞðy (cid:3)~yÞ
r ¼qffiPffiffiffiffiffiffiffiffiffiffiffiffiiffi¼ffiffi1ffiffiffiffiffiffiiffiffiffiffiffiffiffiqi ffiPffiffiffiffiiffiffiffiffiffiffiffiffiffiiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð1Þ
n ðx (cid:3)~xÞ2 n ðy (cid:3)~yÞ2
i¼1 i i i¼1 i i
P P P
n n xy (cid:3) n x n y m x(cid:2) m m
b¼ i¼P1 i i iP¼1 i i¼1 i(cid:4) R(cid:4)100%(cid:5) (cid:4) R(cid:4)100%¼R(cid:4) R(cid:4)100% ð2Þ
n n y2(cid:3) n y2 m (cid:2)y m m
i¼1 i i¼1 i j j j
P
n x m
R%¼Pi¼1 i(cid:4) R(cid:4)100% ð3Þ
n y m
i¼1 i j
(cid:3) (cid:3) (cid:3) (cid:3) (cid:3) (cid:3)
α ¼(cid:3)(cid:3)(cid:3)a(cid:3)(cid:3)(cid:3)¼(cid:3)(cid:3)(cid:3)R(cid:3)1(cid:3)(cid:3)(cid:3)¼(cid:3)(cid:3)e(cid:3)(cid:3) ð4Þ
by(cid:2) b b
MaterialsandMethods
Reagentsandchemicals
Acetonitrile(HPLCgrade)waspurchasedfromYuwangIndustryCo.,Ltd(Shandong,China).
Phosphoricacid(HPLCgrade)wasobtainedfromKermelChemistryReagentCo.,Ltd(Tian-
jin,China)andanhydrousethanol(HPLCgrade)fromFuyuFineChemicalCo.,Ltd(Tianjin,
China).De-ionizedwaterandotherreagentswereofanalyticalgrade.Atotalof27batchesof
ASFsamples(S1-S27)wereselfextractedinlaboratory.Thereferencesample(RS)ofASF
(BatchNo.20151101)wasprovidedbyGuangxiHuaHongPharmaceuticalCo.Ltd.Matrine
(MT,BatchNo.MUST-13021904,purity>99.2%)andOxymatrine(OMT,BatchNo.MUST-
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LinearQuantitativeProfilingMethodMonitorsAlkaloidsofSophoraFlavescens
Fig1.Thestructuresofthemarkercompounds.
doi:10.1371/journal.pone.0161146.g001
13021902,purity>99.5%)wereprovidedbyChengduManSiteBiologicalTechnologyCo.Ltd.
(ChengduInstituteofbiological,ChineseAcademyofSciences).Sophoridine(SPR,batch
No.141029,purity>98%)waspurchasedfromShanghaiWinherbScienceandTechnologyInc.
(Shanghai,China).ThestructuresofthethreemarkercompoundsareshowninFig1.
ExtractionprocedureforAlkaloidsofSophoraflavescenssamples
ThedriedrootofSophoraflavescensAit.waspulverizedintopowder,andthenacidicwater
waspercolatedintoeachpowdersample.ThepercolatingliquidwasconcentratedandthepH
wasadjustedtobetween10and11withabase;then,theliquidwasextractedusingdichloro-
methane.Theextractedliquidwasconcentratedbyrecoveringthedichloromethane,andthe
residuewasdissolvedinethanol.ASFsampleswereobtainedafterevaporationoftheethanol.
Instrumentsandchromatographicconditions
ChromatographicanalysiswasperformedonanAgilent1100HPLCseries(AgilentTechnol-
ogy,USA),equippedwithadiodearraydetector,alowpressuremixquaternarypump,an
onlinedegasserandanautosampler.DataacquisitionwascontrolledbytheChemStation
workstation(AgilentTechnology).ThechromatographicseparationwascarriedoutonanAgi-
lentZOBAXNH column(250×4.6mm,5.0μm)thermostatedat35°C.Themobilephase
2
wascomposedofacetonitrile,anhydrousethanolandwater(82:10:8,v/v/v,containing0.24%
phosphoricacid).Isocraticelutionwasemployedattheflowrateof1.0mL/min.Theinjection
volumewassetat20μL.Thedetectionwavelengthwassetat210nm.
Chromatographicfingerprintswereprocessedbyanin-housedevelopedsoftware,Digitized
EvaluationSystemforSuper-InformationCharacteristicsofTCMChromatographicFinger-
prints4.0(SoftwarecertificatedNO.0407573,China).SPSS16.0andSIMCA13.0werealso
usedfordataanalysis.
Sampleandstandardsolutionpreparation
Stockstandardsolutionswerepreparedbyaccuratelyweighing9.0mg,8.0mgand8.0mgof
MT,OMTandSPRstandardsintoseparatevolumetricflasksof50ml,50ml,25mlrespec-
tively.Thereferencestandardwasdissolvedinadequateethanolandthendilutedtovolume
withethanolandstoredat4°Cforsubsequentuse.Themixedstandardsolutionwasprepared
bypipetting1.0ml,0.5mland1.0mLoftheMT,OMTandSPRstockstandardintoa50ml
volumetricflaskandthendilutingtovolumewithethanol.
Approximately0.10goftheASFsamplewasweighedintoaconicalflask,and25.0mLof
ethanolwasaddedtotheflask.Aftertheconicalflaskwascapped,thewholeflaskwiththecon-
tentwasaccuratelyweighed.Thentheflaskwassonicatedfor15minutes(power320w,fre-
quency40KHZ).Aftercooling,theflaskwasweighedagainandanylostethanolwas
replenished.Afterfiltration,1.0mLofthefiltratewaspipettedintoa50mLvolumetricflask
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LinearQuantitativeProfilingMethodMonitorsAlkaloidsofSophoraFlavescens
Fig2.RepresentativechromatogramsofanASFsample(A),themixedstandards(B)andthe3Dspectrum(C).Theon-lineUVspectraofMT,SPR
andOMT(a,b,cforstandardanda1,b1,c1forsample)areshownnexttothechromatographicpeaks.
doi:10.1371/journal.pone.0161146.g002
anddilutedtovolumewiththemobilephase.Thesamplesolutionwasfilteredthrough
0.45μmMilliporefilterspriortoHPLCanalysis.
ResultsandDiscussion
Quantitationofthethreemarkercompounds
Methodvalidationofquantitativeanalysis. Reversed-phaseliquidchromatography
(RPLC)istypicallyusedfortheanalysisofASF.However,quinolizidinealkaloids(e.g.,MT,
SPRandOMT)areverypolarespeciallywhentheyareprotonated.Thisleadstoinsufficient
retentionandpeaktailingontheRPLCcolumns.Thepresentstudyemployedanaminocol-
umntoseparatethechemicalcomponentsinASFinthehydrophilicinteractionchromatogra-
phy(HILIC)mode.AsshowninFig2,thethreemarkercompoundswerewellretainedand
separatedwithverygoodshape.Inaddition,otherminorcomponentswerealsoelutedmostly
earlyinthechromatogram.TheassignmentofMT,SPRandOMTwascarriedoutbycompar-
ingtheretentiontimesandon-lineUVspectrawiththoseofstandards.
TheHPLCmethodwasvalidatedforlinearity,systemrepeatability,accuracy,limitofdetec-
tion(LOD)andlimitofquantitation(LOQ)insupportofitsapplicationtoquantitativeanaly-
sisofthethreemarkercompounds.ThelinearityoftheHPLCmethodwasassessedatsix
concentrationlevelsofthethreemarkercompoundsasdescribedinTable2.Thecalibration
Table2. Correlationcoefficient(r),linearrange,LODandLOQforthetri-markercompounds.
Compound Regressioneqution r LODb/ng LOQc/ng Linearrange/μg
MT y=1256.8x-12.181a 0.999998 2.1 6.3 0.225~1.800
SPR y=1240.0x-17.252 0.999993 3.3 10.0 0.200~1.600
OMT y=1124.5x-3.042 0.999991 0.83 2.5 0.090~0.720
ayandxwere,respectively,thepeakareasandmasses(μg)oftheanalytes.
bLOD:limitofdetection(S/N=3)
cLOQ:limitofquantitation(S/N=10)
doi:10.1371/journal.pone.0161146.t002
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LinearQuantitativeProfilingMethodMonitorsAlkaloidsofSophoraFlavescens
Table3. Theresultsofthequantitativeanalysisforthetri-markercompoundsandthefingerprintanalysisassessedbyLQPM.
No. Content(mg(cid:2)g-1) P % R% ΔE r b ΔE ΔE α Grade Quality
3C 1 2 3
MT SPR OMT SUM
S1 217.57 231.16 88.55 537.28 96.7 95.5 -1.2 0.997 94.9 -1.9 -0.7 0.007 2 better
S2 195.11 201.96 75.97 473.04 84.7 86.6 1.9 1.000 86.1 1.4 -0.5 0.006 3 good
S3 235.51 248.77 91.73 576.01 102.9 103.2 0.3 1.000 102.9 0.0 -0.3 0.003 1 best
S4 236.03 245.87 90.70 572.59 102.2 102.7 0.5 1.000 102.6 0.4 -0.1 0.000 1 best
S5 232.14 243.02 89.59 564.75 100.8 103.4 2.6 1.000 102.2 1.4 -1.3 0.012 1 best
S6 254.64 263.75 96.80 615.19 109.7 110.5 0.8 1.000 110.2 0.5 -0.4 0.003 3 good
S7 234.36 242.98 94.24 571.58 102.9 103.7 0.8 1.000 102.4 -0.5 -1.3 0.013 1 best
S8 240.58 249.81 92.05 582.44 103.9 104.8 0.9 1.000 104.2 0.3 -0.7 0.006 1 best
S9 257.31 266.62 98.57 622.51 111.1 111.9 0.8 1.000 111.0 -0.1 -1.0 0.009 3 good
S10 244.63 256.93 92.02 593.59 105.5 105.3 -0.2 1.000 105.3 -0.3 0.0 0.000 2 better
S11 237.67 246.59 92.77 577.03 103.3 103.5 0.2 1.000 103.6 0.3 0.1 0.001 1 best
S12 259.75 271.07 101.86 632.67 113.3 113.7 0.4 1.000 112.6 -0.7 -1.1 0.010 3 good
S13 251.95 264.00 94.70 610.65 108.5 107.4 -1.1 1.000 107.2 -1.3 -0.2 0.002 2 better
S14 261.54 271.06 104.91 637.51 114.7 113.7 -1.0 1.000 112.6 -2.1 -1.1 0.009 3 good
S15 221.87 230.42 84.41 536.70 95.7 97.4 1.7 1.000 96.2 0.5 -1.1 0.012 1 best
S16 250.64 260.95 98.17 609.76 109.2 106.9 -2.3 1.000 107.0 -2.3 0.0 0.000 2 better
S17 191.30 234.39 85.53 511.22 92.2 92.1 -0.1 0.996 91.6 -0.6 -0.6 0.006 2 better
S18 217.96 269.92 97.40 585.28 105.4 105.0 -0.4 0.996 104.4 -1.0 -0.5 0.005 1 best
S19 180.07 173.33 63.34 416.74 73.8 75.1 1.3 0.998 76.5 2.7 1.4 0.018 4 fine
S20 255.65 244.82 92.22 592.70 105.5 103.3 -2.2 0.998 104.1 -1.4 0.7 0.007 1 best
S21 245.99 260.49 98.66 605.14 108.6 104.3 -4.3 0.999 106.0 -2.6 1.7 0.017 2 better
S22 231.75 243.71 90.84 566.30 101.3 101.5 0.2 0.999 103.8 2.5 2.3 0.022 1 best
S23 220.09 231.14 86.29 537.52 96.2 93.0 -3.2 0.999 94.2 -2.0 1.1 0.012 2 better
S24 211.85 223.33 83.54 518.72 92.9 91.9 -1.0 0.999 93.0 0.1 1.0 0.011 2 better
S25 215.84 225.80 86.72 528.36 95.0 92.7 -2.3 0.998 92.8 -2.2 0.2 0.001 2 better
S26 202.66 211.18 78.75 492.59 88.1 84.6 -3.5 0.998 85.7 -2.4 1.1 0.012 3 good
S27 197.52 218.35 78.83 494.70 88.4 88.5 0.1 0.998 89.3 0.9 0.8 0.009 3 good
RFP 228.77 240.79 89.50 559.06 100.0 100.0 0.0 1.000 100.0 0.0 0.0 0.000 1 best
RS 228.63 237.62 86.59 552.84 98.5 98.8 0.3 0.998 99.3 0.8 0.5 0.005 1 best
doi:10.1371/journal.pone.0161146.t003
curveswereestablishedbyplottingthepeakareaversustheinjectedmass(μg)ofthestandard
markercompounds.Acceptablelinearitywasdemonstratedforthethreemarkercompounds
intheconcentrationrangesuitableforsampleanalysisasshowninTable2.Systemrepeatabil-
itywasevaluatedbythepeakareaofthethreemarkercompoundsfollowingconsecutiveinjec-
tionsofthemixedstandardsolution.Therelativestandarddeviation(RSD)wasfoundnotto
exceed0.62,1.15and1.10%(n=6)forMT,OMTandSPR,respectively.Theaccuracyofthe
HPLCmethodwasdeterminedbyrecoveryusingthestandardadditionmethod.Themean
recoveryofthethreemarkercompoundswasbetween98.4%and100.1%,suggestingaccept-
ableaccuracyofthemethod.TheLODandLOQweredeterminedbyappropriatelydiluting
themixedstandardsolutionsandtheresultsaresummarizedinTable2.
Sampleanalysis. ThecontentofMT,OMTandSPRinASFsamplesandonereference
sample(RS)wasdeterminedusingthevalidatedHPLCmethod,andtheresultsarereportedin
milligramspergramofASFinTable3.Somevariationinthecontentofeachalkaloidwas
observedinthesamplesthatweretested.Forexample,thehighestcontentsofMT,SPR,OMT
andthetotalamount(SUM)ofthemwereinS14,meanwhile,thelowestcontentsofMT,SPR,
OMTandthesumofthemwereinS19.
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Fig3.ThePCAloadingplot(A)andscoresscatterplot(B)foralltheASFsamples.
doi:10.1371/journal.pone.0161146.g003
Inordertoevaluatethediscriminatingabilityofthemarkercompounds,principlecompo-
nentanalysis(PCA)wasperformedusingtheindividualcontentsofthemarkercompounds
andthetotalamount(SUM)astheinputdatatoconstructatwo-dimensionalmatrixwith29
observationsand4variables(29x4).Asshownintheloadingplot(Fig3A),allfourvariables
arepositivelycorrelatedtoPC1withthesumofthreemarkercompoundshavingthehighest
loadingonPC1.Incontrast,onlyMTispositivelycorrelatedtoPC2significantlyandOMT
andSPRhavenegativeloadingsonPC2.Thetwo-componentPCAmodelaccountsfor95.40%
and3.94%ofthevariationinPC1andPC2,respectively.
Thescorescatterplot(Fig3B)showsthatallthesamplesaregroupedintotwoclusters
markedbyGroup1andGroup2,respectively.ThesamplesinGroup1(S1,S2,S15,S17,S19,
S23,S24,S25,S26andS27)allhavenegativevaluesonPC1,indicatingthatthecontentofthe
markercompoundswereallrelativelylower(intherangesof180.07–221.87mg/gforMT,
173.33–234.39mg/gforSPR,63.34–88.55mg/gforOMTand416.74–537.52mg/gforSUM,
respectively).ThesamplesinGroup2(S3-S14,S16,S18,S20,S21andS22)withpositiveval-
uesonPC1haverelativelyhighercontentofthemarkercompounds(intherangesof217.96–
261.54mg/gforMT,243.02–271.07mg/gforSPR,89.59–104.91mg/gforOMTand564.75–
637.51mg/gforSUM,respectively).Among27samples,onlythreesamples(S17,S18and
S19)werefoundtobeoutliers(outsidetheellipse)basedonthecontentofthemarkercom-
pounds.Itisalsointerestingtonotethatthereferencesample(RS)withanegativevalueon
PC1andthereferencefingerprint(RFP)aresituatedveryclosetotheorigin,indicatingthe
“synthesized”referencefingerprint(RFP)isverysimilartoanindependentlyacquiredrefer-
encesample.
Chromatographicfingerprintanalysis
Methodvalidationoffingerprintanalysis. ThepeakofMTwasassignedandselectedas
thereferencepeak(showninFig2),thentherelativeretentiontimeandtherelativepeakarea
canbecalculatedout.Theinstrumentprecisionwas6replicatedloadingS1samplesolutionto
determinetheaveragequalitativesimilarity(AQLS)of0.963(RSD=3.5%)andtheaverage
quantitativesimilarity(AQTS)of100.5%(RSD=0.73%).Thesamplesolutionstabilitywas
analyzedonceevery95minutesafterpreparedwithin11hoursandwasfoundstablewith
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LinearQuantitativeProfilingMethodMonitorsAlkaloidsofSophoraFlavescens
Fig4.TheHPLCfingerprintsof27batchesoftheASFsamples,thereferencefingerprint(RFP),andthereferencesample(RS)detectedat210nm.
(A)NormalHPLCfingerprints(B)therelativecharacteristicprofilingsreferencedbypeakMT.
doi:10.1371/journal.pone.0161146.g004
AQLSof0.978(RSD=1.75%)andAQTSof100.2%(RSD=1.31%).Themethodrepeatability
wasassessedbyanalyzingsixindependentlypreparedsamples(S1)byaboveanalyticalproce-
durestogiveAQLSof0.976(RSD=0.75%)andAQTSof100.1%(RSD=0.83%).Theabove
AQLSandAQTSwerecalculatedoutbytheself-madeTCMfingerprintsoftware.Thevalida-
tionresultsexhibitedthatthemethodsatisfiedthefingerprintanalysiscriteria.
EvaluatingASFqualitybyLQPMbasedonthefingerprintprofiling. Thevalidated
HPLCmethodwasusedtogeneratechromatographicfingerprintsoftheASFsamples(shown
inFig4A).Attheanalyticalwavelengthof210nm,18commonpeakswerefoundinallsam-
plesandtheamplifiedchromatogramwasgivenwithlabeledpeaknumbersinFig2A.Theref-
erencefingerprint(RFP)wasconstructedastheauthenticallyfingerprintbyaveragingallthe
27samplefingerprints.Therelativecharacteristicfingerprint(RCFP,showninFig4B)was
constructedbyplottingtherelativepeakareaversustherelativeretentiontime.Thesamplefin-
gerprintsandreferencefingerprintwereimportedtothein-housesoftwaretocalculatethe
assessingresultsaspresentedinTable3.Theresultsshowthatallthesampleshavelinearquali-
tativesimilarity(r)higherthan0.996anderrortermα(cid:7)0.022,indicatingthatallthesamples
aresimilarinthedistributionofchemicalcomponents.Incomparison,thelinearquantitative
similarity(b)hasawiderrange(76.5–112.6%).AsaLQTSmeasure,bcanexactlydiscriminate
thesamplesfromtotalcontentsofallfingerprintpeaksbutactuallyrisdisabledforthefunc-
tion.Forexample,S19haslowerlinearquantitativesimilarity(b)(76.5%),althoughthelinear
qualitativesimilarity(r)isveryhigh(0.998).Thisindicatethatbisausefuldiscriminatingtool
todifferentiatetheASFsampleswhenrisveryclosetounity.
Intermsofthecriteria(showninTable1),thequalitiesofS3,S4,S5,S7,S8,S11,S15,S18,
S20,S22andRSwerebest(Grade1),thoseofS1,S10,S13,S16,S17,S21,S23,S24andS25were
better(Grade2),andthoseofS2,S6,S9,S12,S14,S26andS27weregood(Grade3),exceptfor
thatofS19asfine(Grade4)duetothemuchlowercontentsforthe18components.Incom-
parison,thePCAmethodidentifiedthreesamples(S17,S18andS19)asoutliers,andS18was
singledoutbecausetherelativeamountsofthemarkercompoundsaremuchhigherthanother
samples.However,itdoesnotnecessarilymeanthatS18haspoorquality.Infact,S18has
acceptablequality(Grade1)basedonthequantitativefingerprintanalysis.Inaddition,two
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LinearQuantitativeProfilingMethodMonitorsAlkaloidsofSophoraFlavescens
moresampleswereidentifiedasdissimilar(S17belongedtoGrade2andS19belongedto
Grade4)basedondifferentlinearquantitativesimilarities.
ThisobservationhighlightsthepointthatPCAcanbeoverdiscriminatinginsomecases
andthequantitativefingerprintanalysiscanprovideamoreaccurateevaluationoftheherbal
samples.
InvestigatingthecompositionsimilarityofASFreferencefingerprint. Accordingto
ASFreferencefingerprint,wecancalculatethecompositionsimilarityofthethreebiggest
peaksi.e.peak16,peak13andpeak17with0.478,0.458,0.055ofr,and36.9%,35.8%and
12.6%ofb,respectively.Thetotalvalueofrforthetri-markeris0.99andthetotalvalueofb
forthetri-markeris85.3%,whichclearlyexpressforthedominantcontribution,respectively.
SotheASFqualityisabsolutelyoccupiedbythetri-markerbuttheotherlowercontentsof
componentscanbecontrolledbybfromaquantifyingprofilepattern.IfMTpeakwasselected
asthereferencestandard,wecancalculatetherelativeweightcorrectionfactorSPR/MTas
f ¼ASPRmMT ¼1:024(RSD=0.77%,n=5)andOMT/MTasf ¼AOMTmMT ¼1:118
SPR=MT AMTmSPR OMT=MT AMTmOMT
(RSD=0.48%,n=5)accordingtothelinearregressionequations(seeninTable2)oftri-
marker.BytakingthosetwofactorsintoEqs1and2respectively,wecangetthechangesforr
andbwithin1.2%thatcertainlycanbeignored.
CorrelatingLQTSwiththreemarkerAnalyses
Inthisstudy,thealkaloidcontentintheASFwasaccuratelyquantitatedusingthemarkercom-
pounds(MT,OMTandSPR).However,thequantitativeanalysisrequiresthereferencestan-
dards,calibrationandmoretime.Evenifquantitationisfeasiblewhenthereferencestandards
areavailable,thequantitativeresultsareonlymeaningfulwhenacceptancecriteriaforthespe-
cificcompoundsexist.Unfortunatelythisisnotthecaseformostoftheherbalmedicines.
Therefore,fingerprintanalysisbecomesmorecriticalifitisconsistentwiththequantitative
results.
Asdiscussedin“EvaluatingASFqualitybyLQPMbasedonthefingerprintprofiling”sec-
tion,thelinearquantitativesimilarity(b)isfoundtobemorediscriminatingfortheAlkaloids
ofSophoraflavescens.TherelationshipbetweenLQTS(b)andthequantitativeresultsofthe
markercompoundsisfurtherexplored.First,theweightcontentofeachmarkercompoundin
allthesampleswasaveragedtogenerateitsaveragecontent((cid:2)z),thenthepercentageoftheith
markercontent(Pi%)for27samples(seeninS1Table)wascalculatedout(showninEq5)
usingamasscoefficientfj ¼mmRj.Secondly,themeanofPi%valuesofthethreemarkercom-
poundsineachsample(P3C%)wascalculatedaccordingtoEq6.Meanwhile,R%wascalculated
byEq4.Finally,Pi%,P3C%andR%of27sampleswererespectivelyplottedvs.basshownin
Fig5.LinearregressionshowsthatthecorrelationcoefficientsbetweenPi%andbare0.9471,
0.9664and0.9597forMT,SPRandOMT,respectively.Incomparison,thecorrelationcoeffi-
cientbetweenP3C%andbissignificantlyhigher(0.9884).Thisindicatesthatbishighlycorre-
latedtothecontentofthemarkercompounds.Itisnotsurprisingthatbisalsohighly
correlatedtotheapparentcontentsimilarity(R%)asshowninFig5E,andthecorrelationcoef-
ficientbetweenR%andbreachedthemostexcellentvalueof0.9950.Thisdemonstratesthat
LQTS(b)isareliablesubstituteforthequantitativecontentofthemarkercompoundsandis
veryeffectiveinquantitativelyevaluatingthequalityofherbalmedicines.Therefore,themulti-
plemarkersanalysesforqualitycontrolcanbesubstitutedbyLQPM,whichissimple,briefly,
accurateandeconomic.ASFreferencefingerprintcontained228.77mg/gofMT,240.79mg/g
ofSPRand89.50mg/gofOMT,andthetotalcontentofthetri-markerwas559.06mg/gwhich
wasupto70%(g/g)whenwaterwastakenout(pleaseseethewatercontentinbelowsection
‘OveralldistributionofbasicsubstancesinASFsamples’).Sowecanimmediatelycalculate
PLOSONE|DOI:10.1371/journal.pone.0161146 August16,2016 9/15
LinearQuantitativeProfilingMethodMonitorsAlkaloidsofSophoraFlavescens
Fig5.Thelinearregressionplotsofthecontentpercentage(P%)ofthemarkercompoundsvs.thelinearquantitativesimilarity(b).(A)forMT,(B)
i
forSPR,(C)forOMT,(D)forP %,(E)forR%,(F)forallthemarkercompounds,P %andR%.
3C 3C
doi:10.1371/journal.pone.0161146.g005
eachcontentofthethreemarkercompoundsaccordingtob.FromTable3,thebiggesterror
wasnotmorethan3.5%forΔE1=R%−P3C%,ΔE2=b−P3C%andΔE3=b−R%.Inaword,
LQTS(b)isabetterwaytodeterminetheoverallcontentsofASF.
z
P%¼ if (cid:4)100% ð5Þ
i (cid:2)z j
1X
3
P %¼ P (cid:4)100% ð6Þ
3C 3 i¼1 i
CorrelatingLQTSwithLQLS
Thereasonwhyweusetwosimilarities(LQLSandLQTS)todifferentiateASFqualitybasedon
chromatographicprofilesisthatthereisnocorrelationbetweenLQTSandLQLSof29profiles
inTable3.ThePearsoncorrelationcoefficientR2=0.1194(n=29)reflectsthatwecannot
thoroughlyevaluatequalityofTCMintheaccuratewaybyonlyusingLQLS(rallmorethan
0.996).SoitismuchnecessarytousethesecondmeasureofLQTS(b)anditisthehighestlevel
evaluationforassessingfingerprintprofilesofTCM.Fig6nearlydisplaysanorthogonalitycor-
relationbetweenrandb,whichindicatesthattheLQTSassessismoreimportantthanLQLS.
ThereforeLQPMisanovelfingerprintassessingmethodlikeSQFM[37],inwhichbothLQLS
andLQTSareusedtogetherforevaluatingTCMquality,andastatisticalerrorisusedtomoni-
torthemethoditself.
PLOSONE|DOI:10.1371/journal.pone.0161146 August16,2016 10/15