Simultaneous Determination of Eight Alkaloids in Rat Plasma by UHPLC-MS/MS after Oral

15 Pages·2016·2.55 MB·English

molecules
Article
Simultaneous Determination of Eight Alkaloids in
Rat Plasma by UHPLC-MS/MS after Oral
Administration of Coptis deltoidea C. Y. Cheng et Hsiao
and Coptis chinensis Franch
LuLiu1,Zhi-BinWang2,YangSong3,JingYang4,Li-JunWu1,Bing-YouYang2,
Qiu-HongWang2,Li-QianWang1,Ru-XuanWang1andChun-JuanYang1,*
1 DepartmentofPharmaceuticalAnalysisandAnalyticalChemistry,CollegeofPharmacy,
HarbinMedicalUniversity,No.157BaojianRoad,NangangDistrict,Harbin150081,China;
[email protected](L.L.);[email protected](L.-J.W.);[email protected](L.-Q.W.);
[email protected](R.-X.W.)
2 KeyLaboratoryofChineseMateriaMedica(MinistryofEducation),
HeilongjiangUniversityofChineseMedicine,24HepingRoad,XiangfangDistrict,Harbin150040,China;
[email protected](Z.-B.W.);[email protected](B.-Y.Y.);[email protected](Q.-H.W.)
3 SchoolofPharmacy,ChinaMedicalUniversity,Shenyang110001,China;[email protected]
4 AnalyticalDepartment,Johnson&Johnson,199GrandviewRoad,Skillman,NJ08558,USA;
[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:ThomasJ.Schmidt
Received:9May2016;Accepted:8July2016;Published:14July2016
Abstract: Aultra-highperformanceliquidchromatography-electrosprayionizationtandemmass
spectrometry (UHPLC-ESI-MS/MS) method was successfully developed and validated for the
identification and determination of eight alkaloids: tetrahydropalmatine (A); palmatine (B);
magnoflorine(C);columbamine(D);berberine(E);worenine(F);berberrubine(G)andcoptisine(H)
inratplasma,whicharetheactivecomponentsinCoptisdeltoideaC.Y.chengetHsiao(CCY)and
CoptischinensisFranch(CF).Thechromatographicseparationofanalyteswassuccessfullyachieved
onanAgilentSB-C column(1.8µm,150mmˆ2.1mm)usingaprogrammewithamobilephase
18
consisting of acetonitrile and water containing 0.3% acetic acid at a flow rate of 0.25 mL/min.
TheanalytesweredetectedwithatriplequadrupoletandemMSinmultiplereactionmonitoring
(MRM)modeandanelectrosprayionization(ESI)sourceinpositivemode. Thevalidatedmethod
showedgoodlinearityoverawideconcentrationrange(r2>0.991),andlowerlimitsofquantification
(LLOQ) less than 1.1 ng/mL for all analytes, and matrix effects ranged from 85.2% to 106.8%.
Themeanextractionrecoverieswerenolessthan86.4%,andtheprecisionandaccuracywerewithin
the acceptable limits. All analytes were proven to be stable during sample storage and analysis
procedures. Themethodvalidationresultsdemonstratedthattheproposedmethodwassensitive,
specific,andreliable,whichcouldlayafoundationforthepharmacokineticstudyofeightanalytes
afteroraladministrationofCCYandCFinsubsequentstudies.
Keywords: alkaloids;UHPLC-MS/MS;methoddevelopment;ratplasma
1. Introduction
Rhizoma Coptidis (RC), the dried rhizome of Coptis deltoidea C. Y. Cheng et Hsiao (CCY),
CoptischinensisFranch(CF),orCoptisteetaWall,thespeciesinthegenusCoptis(familyRanunculaceae),
has been used in China for thousands of years [1] and is recorded with the Chinese name of
“Huang Lian” in more than thirty classical Chinese medicine books [2]. In Traditional Chinese
Molecules2016,21,913;doi:10.3390/molecules21070913 www.mdpi.com/journal/molecules
Molecules2016,21,913 2of15
Medicine(TCM)itcould“clearthedamp-heat,quenchthefire,andcounteractthepoison”[3],while
recentstudieshaveindicatedthatRCcanbeusedtotreathypertensionandhyperlipidemia,dysentery
Molecules 2016, 21, 913 2 of 14
andgastroenteritis,liverandhepatobiliarydiseases,etc.[4,5]CCY,commonlyknownas“YaLian”,
mayhacovuelda “loclweaerr tyheie dldamdpu-ehetoat,t hqeuelnocnhg tghero fwiret,h apnder cioodunatenrdacot tthhee rppohisyonsi”o [l3o]g, iwcahlilceh raercaenctte srtiusdtiicess , make
itscarcheav[6e ].inCdFicaotfetde nthraetf eRrCre dcatno baes u”Wsede itLo iatrne”at, hhayspebreteennsimono raendw hidyepleyrliupsidedemaisa,t hdeysmenatjeoryr ianngdr edient
inmangyasCtroheinnteesreitihs,e lrivbearl afnodr mheuplaatosbsiuliacrhy adsiseoarsaels,l ieqtcu. i[d4,5“]s ChCuYan, cgomhumaonnglyl ikanno”w[n7 ]a.s T“Yhais Lpialna”n, tmiasym ainly
have a lower yield due to the long growth period and other physiological characteristics, make it scarce
producedintheChongqing,Hubei,Guizhou,ShanxiprovincesofChina. Furthermore,differences
[6]. CF often referred to as ”Wei Lian”, has been more widely used as the major ingredient in many
inkindsandcontentofalkaloidcomponentsexistbetweenCCYandCF[8],whicharecausedbythe
Chinese herbal formulas such as oral liquid “shuang huanglian” [7]. This plant is mainly produced
differencesintheirgrowthconditions,storage,andprocessingoftheherb.
in the Chongqing, Hubei, Guizhou, Shanxi provinces of China. Furthermore, differences in kinds and
Ocnonttheentb oafs ailskaolfoipdr ceovmiopuosnesnttus dexieisst, btehtewemena jCoCrYa catnivd eCcFo [n8]s, twithuiecnh tasrei ncaCusCedY bayn tdheC dFiffaerreenacelks aloids,
includiinn gthteeirtr garhoywdthro cpoanldmitiaotnins,e st(oAra);gpe,a alnmda ptirnocee(sBsi)n;gm oaf gthneo hfleorbri. ne(C);columbamine(D);berberine(E);
worenine(OFn); tbheer bbaesrirsu obf inpreev(Giou)sa nstdudcioesp, ttihsein mea(jHor) a(cFtiivgeu rceon1s)t,iteutecn.tsI ninr eCcCeYn taynde aCrsF, avrae raiolkuaslobidiso,l ogical
activitiinecsluodfintgh etesterahcoydmroppoanlmenattisneh (aAv)e; pbalemeantinree p(Bo)r; tmedag,nsouflcohrinaes (Ca)n; tciomluimcrboabmiainle[ (9D],); baenrtbiecrainnec e(Er);[ 10,11],
worenine (F); berberrubine (G) and coptisine (H) (Figure 1), etc. In recent years, various biological
anti-oxidative[12]andcardioprotective[13]effects,etc. Forexample,berberinesignificantlyreduced
activities of these components have been reported, such as antimicrobial [9], anticancer [10,11],
myocardialischemia/reperfusion-inducedmyocardialinfarctsize,improvedcardiacfunction,and
anti-oxidative [12] and cardioprotective [13] effects, etc. For example, berberine significantly reduced
suppressedmyocardialapoptosisandoxidativedamage[14]. Itisfoundthatfivemajoralkaloidsin
myocardial ischemia/reperfusion-induced myocardial infarct size, improved cardiac function, and
RCpresvupenprteesdsebdo mdyyowcaeridgihalt agpaoipnt,orseisd auncde doxtihdaetisveer udammtaogtea [l1c4h].o Ilte iss tfeoruonld,a tnhadt ifnivcer emaasjeodr atlhkealhoiidgsh i-nd ensity
lipoproRtCei pnrecvheonlteesdt ebroodlyo wfheiagmhts gtaeirns, [r1ed5]u.cAeds tfhoer stehruemo tthotearl achnoalleystteerso,lc, oanpdti isnincree,apseadl mthaet hinigeh,-mdeangsnityo florine
andbelripboeprrroutbeiinn ech,ohlaesvteerboel oefn hraempsoterrtse d[15to]. Ahas vfoer athnet iomthiecrr aonbailaylteosr, caonpttii-siinnfle,a pmalmmaattionrey, mpargonpoeflrotrieinse[ 16,17].
and berberrubine, have been reported to have antimicrobial or anti-inflammatory properties [16,17].
Nevertheless,tothebestofourknowledge,littleresearchhasaddressedthepharmacokineticsofother
Nevertheless, to the best of our knowledge, little research has addressed the pharmacokinetics of
alkaloids including magnoflorine, worenine, berberrubine from CCY and CF, which also showed
other alkaloids including magnoflorine, worenine, berberrubine from CCY and CF, which also
a wide range of useful biological activities, such as antifungal [18], anti-inflammatory [19], and
showed a wide range of useful biological activities, such as antifungal [18], anti-inflammatory [19],
antitumoractivities[20].
and antitumor activities [20].

Figure1.Cont.
Molecules2016,21,913 3of15
Molecules 2016, 21, 913 3 of 14
Figure 1. MS/MS fragmentation patterns of tetrahydropalmatine (A); palmatine (B); magnoflorine (C);
Figure1.MS/MSfragmentationpatternsoftetrahydropalmatine(A);palmatine(B);magnoflorine(C);
columbamine (D); berberine (E); worenine (F); berberrubine (G); coptisine (H); bifendate (I.S.).
columbamine(D);berberine(E);worenine(F);berberrubine(G);coptisine(H);bifendate(I.S.).
Earlier publications have described methods, including high-performance liquid chromatography
Eawrlitihe rupltruab-vliicoaletti odnetsechtaiovne [2d1e]s, hcrigibhe-pdermfoertmhaondcse, liiqnucildu cdhirnogmhaitgoghr-appehryfo wrimtha DnAceDl diqeuteicdtiocnh r[2o2m], aatnodg raphy
withulhtrigah-v-pioerlfeotrdmeatneccet iloiqnu[i2d1 c]h,rhoimgha-topgerrafpohrym-ealneccterolsiqpuraiyd icohnrizoamtioanto-tgarnadpemhy mwaistsh sDpeActDrodmeetteryct i[o23n,2[42]2 ],and
that have been used in the qualitative or quantitative analysis of alkaloids from the genus Coptis, single
high-performanceliquidchromatography-electrosprayionization-tandemmassspectrometry[23,24]
herb or herbs used together in couples (Coptis-Evodia herb couple [25], scutellaria-coptis herb couple
that have been used in the qualitative or quantitative analysis of alkaloids from the genus Coptis,
[26]), at most six or seven alkaloids simultaneously. However, to date, there are few publications
singleherborherbsusedtogetherincouples(Coptis-Evodiaherbcouple[25],scutellaria-coptisherb
concerning CCY, especially the pharmacokinetics. Therefore, it is of great importance to develop a
couples[p2e6c]i)f,ica, tsemnosistitvsei xanodr ssiemvpelne amlketahloodid tos ssiimmuullttaanneeoouuslsyl yd.etHeromwineev tehre, tmoadina tceo,mthpeorneeanrtse offe CwCpYu abnldic ations
concerCnFin ign bCioClYog,iceaslp seacmiapllleys wthheicph hcaorumlda bceo akpinpleietidc sin. cTohmepraerfeo rthee, ipthiasrmofacgorkeiantetiimc cphoarratcatnerciestitcos odfe velop
aspeciCfiCc,Ys aennds iCtiFv ien afonldlowsi-mupp sletumdieest.h odtosimultaneouslydeterminethemaincomponentsofCCY
andCFinbInio ltohigsi csatuldsay,m ap lreaspiwd haincdh cseonuslidtivbee haipghp-lpieedrfoinrmcaonmcep alirqeuitdh echprhoamramtoagcroakpihnye-etilcecctrhoasrparcatye ristics
ionization tandem mass spectrometry was developed for the simultaneous determination of eight
ofCCYandCFinfollow-upstudies.
alkaloids in rat plasma. The method we developed should be useful in pharmacokinetics studies after
In this study, a rapid and sensitive high-performance liquid chromatography-electrospray
oral administration of CCY and CF which could provide important and valuable information for
ionizationtandemmassspectrometrywasdevelopedforthesimultaneousdeterminationofeight
improving RC clinical therapeutic efficacy as well as a better understanding of the differences and
alkaloiedffsiciancyr aotf tphleassem twao. dTrhugesm. Tehtihs owdillw bee tdhee vneexlot ppreodgrsahmo oufl doubr ereusesaerfcuhl teinamp. harmacokinetics studies
afteroraladministrationofCCYandCFwhichcouldprovideimportantandvaluableinformation
forimp2r. oRveisnugltsR aCndc lDinisiccaulssthioenr apeuticefficacyaswellasabetterunderstandingofthedifferencesand
efficacyofthesetwodrugs. Thiswillbethenextprogramofourresearchteam.
2.1. Method Development
2. ResultsandDiscussion
2.1.1. UHPLC-MS/MS Optimization
2.1. MethodADlkeavleoliodpsm haendt stronger signal responses in the positive ion mode compared to the negative one.
The MS/MS ion-transitions were monitored in MRM mode to increase the specificity of the detection
2.1.1. UmHetPhLodC -tMo mS/eMet SthOe pretqimuiirzeamteionnts of the complexity of biological samples. In the total ionization
chromatography (TIC) spectra, the analytes formed predominantly protonated molecules at m/z 356.0
Aflokra tleotirdashyhdardopsatlrmonatgineer (sAig),n aat lmr/ezs 3p4o2n.2s feosr imnatghneopfloorsiintiev (eC)i,o ant mm/oz d33e9c.2o mforp caorleudmtboamthineen (eDg)a, taitv eone.
TheMSm//zM 3S22i.o2 nfo-trr baenrsbietirorunbsinwe e(rGe),m aot nmi/tzo 4r1e8d.9i nfoMr bRifMendmatoed (eI.Sto.). iWnchrielea,s peatlhmeastipnee c(iBfi)c, ibteyrboefrtinhee (dEe),t ection
methodwotroenmineee (tFt)h aendre cqoupitriseimnee (nHts) porfedthoemcinoamntplyl efxoirtmyeod fqbuiaotelorngaircya lamsammopnliuesm. iIonnst h[Me]t+o atat l35io2.n2i, zation
chroma3t3o6g.2r,a 3p34h.y2 a(TndIC 3)20s.p0 einct trhae, TthICe sapneacltyrate. Fsifgourrme 1e dshpowresd tohme iionna pnatilrys pofr othteo neiagthetd anmaloylteecsu alneds tahtem I.S/.z 356.0
fortetr ahydropalmatine(A),atm/z342.2formagnoflorine(C),atm/z339.2forcolumbamine(D),
atm/z322.2forberberrubine(G),atm/z418.9forbifendate(I.S.). While,palmatine(B),berberine(E),
worenine (F) and coptisine (H) predominantly formed quaternary ammonium ions [M]+ at 352.2,
336.2,334.2and320.0intheTICspectra. Figure1showstheionpairsoftheeightanalytesandthe
Molecules2016,21,913 4of15
I.S.Inordertomakethisanalyticalmethodmoreaccurate,weusedthestandardoftheanalytesand
theinternalstandard(I.S.)tosearchforthequalifierions. Toobtainthemaximumsensitivityofthe
MRM,parameterssuchasfragmentor,collisionenergyandthenitrogenflowratewereoptimized.
ParametersmentionedabovewerelistedinTable1.
Table1.Quantitative,QualifierionsandMSparametersofeightalkaloidsandI.S.
Compounds IonPair(m/z) QualifierIon(m/z) Fragmentor(V) CollisionEnergy(V) Polarity1
Bifendate(I.S.) 418.9Ñ342.8 284.8 78 18 Positive
Tetrahydropalmatine 356.0Ñ192.0 165.0 159 27 Positive
Palmatine 352.2Ñ336.2 308.2 158 30 Positive
Magnoflorine 342.2Ñ265.1 58.2 134 22 Positive
Columbamine 339.2Ñ323.2 295.1 160 29 Positive
Berberine 336.2Ñ320.1 292.2 136 30 Positive
Worenine 334.2Ñ261.1 233.0 181 49 Positive
Berberrubine 322.2Ñ307.2 279.2 160 29 Positive
Coptisine 320.0Ñ262.0 292.2 167 29 Positive
1Thecellacceleratorvoltagevaluedoftheseeightcompoundsare5V.
ThecomplexityofthechemicalconstituentsofTCMmayinfluencethequalitativeandquantitative
analysisoftargetcompounds,soweuseda15cmAgilentSB-C column(1.8µm,150mmˆ2.1mm)
18
to avoid any crosstalk of the interfering constituents. Various ratios of organic phase (methanol,
acetonitrile)towaterphaseinclusionofpHmodifiers(ammoniumacetate,formicacid,ammonia)
weretestedtoobtaingoodseparationsandidealpeakshapes. Themobilephaseconsistingofa0.3%
aceticacidaqueoussolution(A)andacetonitrile(B)wasrunataflowrateof0.25mL/minaccording
tothegradientelutionprogramasshowninTable2. Thecolumntemperaturewassetat40˝Candthe
injectionvolumewas10µL.
Table2.Gradientelutionprogramofmobilephase.
Time(min) A% B%
0 65 35
0–2 45 55
2–3 38 62
3–4.5 38 62
4.5–7.0 30 70
7.0–7.5 65 35
2.1.2. SelectionoftheInternalStandardandtheExtractionMethod
Compared with some compounds (such as theophylline, phenacetin, etc.) we tested, the I.S.
bifendatehadamoresuitableretentiontime,agoodpeakshapeinthechromatographicseparation.
Extractionmethodsincludingliquid–liquidextraction(LLE)andproteinprecipitationwithmethanol,
acetonitrileweretestedduringthesamplepreparation. Forproteinprecipitation,thelowerrecovery
andobviousmatrixeffecthinderedfurtherdevelopment. Moreover,amongalltheextractionsolvents
tested(ethylacetate,diethylether,chloroform,acetone[27]),wefoundthatLLEwithacetoneproduced
thehighestresponseandnegligiblematrixeffect.
2.2. MethodValidation
2.2.1. SelectivityandSpecificity
Figure 2 shows the representative MRM chromatograms of the blank rat plasma (I); a spiked
plasmasamplewiththeanalytesandtheI.S.(lowqualitycontrolsample,LQC,II);aplasmasample
collectedat0.25hafteranoraladministrationofCCYextract(III);aplasmasamplecollectedat0.75h
afteranoraladministrationofCFextract(III).
Molecules2016,21,913 5of15
Molecules 2016, 21, 913 5 of 14
Theresultsshowedthattherewerenoendogenoussubstancepeaksfromtheratplasmasamples
The results showed that there were no endogenous substance peaks from the rat plasma samples
andadnrdu dgrumge mtaebtoalbiotelitpee paekasksin itnetrefreferirningg wwiitthh tthhee aannaallyytetes saanndd thteh eI.SI.. Sa.t athtet hreeterentteionnt itoimnetism. Aens.d Ainn ad in
aLQLCQCsa smamplpel,ec, ocommppoouunndd AA––HH aanndd II..SS.. eelluutteedd aatt 44.3.35566, ,55.1.1494,9 2,.28.9839,3 4,.428.278, 75,.358.32,8 25.,051.40,1 44.,8047.8, 047.6,142.6, 12,
7.0578.0m58in m,rine,s preescpteivcteilvye.lyT. hTehae naanlayltyetsesc ocouuldldb bee eeaassiillyy ddiiffffeerreennttiiaatetedd frforomm thteh erarta ptlpaslamsam maamtraixt raixnda nd
quanqtuiatanttiivtaetliyvedlye tdeermterimneidneadt atht tehleo lwowererli lmimitito offq quuaannttiifificcaattiioonn ((LLLLOOQQ)) lelevveel.l .

Figure2.Cont.
Molecules2016,21,913 6of15
Molecules 2016, 21, 913 6 of 14

Figure 2. Typical MRM chromatograms of tetrahydropalmatine(A); palmatine (B); magnoflorine (C);
Figure2.TypicalMRMchromatogramsoftetrahydropalmatine(A);palmatine(B);magnoflorine(C);
columbamine (D); berberine (E); worenine (F); berberrubine (G); coptisine (H); bifendate (I.S.) in rat
columbamine(D);berberine(E);worenine(F);berberrubine(G);coptisine(H);bifendate(I.S.)inrat
plasma: (I) Blank rat plasma; (II) Blank plasma spiked with eight alkaloids and I.S.; (III) a plasma
plasma: (I)Blankratplasma;(II)BlankplasmaspikedwitheightalkaloidsandI.S.;(III)aplasma
sample from a rat 0.25 h after oral administration of Coptis deltoidea C. Y. cheng et Hsiao (CCY) extract;
samplefromarat0.25hafteroraladministrationofCoptisdeltoideaC.Y.chengetHsiao(CCY)extract;
(IV) a plasma sample from a rat 0.75 h after oral administration of Coptis chinensis Franch (CF) extract.
(IV)aplasmasamplefromarat0.75hafteroraladministrationofCoptischinensisFranch(CF)extract.
2.2.2. Linearity and Lower Limit of Quantification
2.2.2. LinearityandLowerLimitofQuantification
The calibration curve, correlation coefficient (r2), linear range, and LLOQ of each component are
shTohwenc ianl iTbarbaltei o3n. Tchuer lvoew,ecsot rcroenlacetniotrnatcioonesf fiwciitehn RtS(Dr2%), l<i n20e%ar wraenreg tea,kaenn dasL LLLOOQQso wf eitahc ah scigonmalp-toon-ent
arensohisoew (Sn/Nin) rTaatibol e> 130., wThhiechl owwaes sstencosinticveen etnraotuigohn sfowr tihthe pRhSaDrm%ac<ok2i0n%eticw seturediteask inen raat splLaLsmOaQ. Aslwl ith
a sicgonrarel-ltaoti-onno cisoeef(fSic/ieNn)tsr eaxticoee>de1d0 ,0.w99h0i5c hwhwicahs isnednisciattievde tehnato euigghht faonratlhyteesp ehxahrimbiatecdo kgionoedt ilcinsetaurditiye. s in
ratplasma. Allcorrelationcoefficientsexceeded0.9905whichindicatedthateightanalytesexhibited
goodlinearity.
Molecules2016,21,913 7of15
Table3.Theregressionequations,linearrangesandLLOQsforthedeterminationoftheanalytesin
ratplasma.
Compounds RegressionEquation R2 LinearRange(ng/mL) LLOQ(ng/mL)
Tetrahydropalmatine Y=15.358X+1.720ˆ10´2 0.9964 0.5–2028 0.5
Palmatine Y=112.57X+5.3976ˆ10´2 0.9928 0.1–428 0.1
Magnoflorine Y=0.3350X+3.272ˆ10´3 0.9906 1.1–4320 1.1
Columbamine Y=2.3498X+5.334ˆ10´2 0.9956 0.6–2230 0.6
Berberine Y=18.189X+0.117ˆ10´1 0.9985 0.1–422 0.1
Worenine Y=2.4691X+3.378ˆ10´3 0.9907 0.6–2220 0.6
Berberrubine Y=12.738X+1.730ˆ10´3 0.9945 1.1–4420 1.1
Coptisine Y=2.5543X+3.665ˆ10´2 0.9905 0.2–800 0.2
2.2.3. PrecisionandAccuracy
Theintra-andinter-dayprecisionandaccuracyofthemethodarepresentedinTable4which
wasevaluatedbymeasuringsixreplicatesofLLOQandqualitycontrolsample(QC)samplesatthree
concentrationlevels(LQC,mediumqualitycontrolsample,MQC,andhighqualitycontrolsample,
HQC) on the same day for three consecutive days. The intra and inter-day precisions (RSD%) of
theseanalyteswerealllessthan14.4%forQCsamplesand18.8%forLLOQ,respectively, andthe
accuracyrangedfrom´14.4%to14.1%. Theresultsindicatedthattheprecisionandaccuracyvalues
wereacceptable.
Table4.Precisionandaccuracyofthedeterminationofeightalkaloidsinratplasma(n=18,6replicates
perdayfor3days).
SpikedConcentration MeasuredCONC Accuracy Intra-Day Inter-Day
Compounds
(ng/mL) (ng/mL) (%) Precision(%) Precision(%)
0.5 0.5˘0.1 ´1.0 17.6 18.8
2.0 2.1˘0.3 ´3.4 13.6 3.7
Tetrahydropalmatine
50.7 53.1˘6.9 ´3.0 12.9 12.9
1622 1759˘172 8.4 9.1 13.8
0.1 0.1˘0.02 8.5 18.5 15.6
0.4 0.5˘0.07 11.2 13.4 11.9
Palmatine
10.7 11.1˘1.5 3.4 14.2 10.3
342 368˘41.8 7.5 11.8 6.7
1.1 1.3˘0.1 14.1 11.7 6.6
4.3 4.3˘1.1 7.1 14.3 4.7
Magnoflorine
108 116˘13.8 7.5 12.0 10.3
3456 3817˘485 10.4 12.5 14.4
0.6 0.6˘0.04 ´14.4 14.9 14.2
2.2 2.5˘0.3 9.8 14.3 3.5
Columbamine
55.8 53.9˘6.7 ´3.4 12.3 13.5
1784 1713˘193 ´4.0 11.0 13.4
0.1 0.1˘0.01 ´2.9 10.0 17.8
0.4 0.5˘0.1 5.5 14.3 6.3
Berberine
11.1 10.7˘1.0 ´7.3 8.7 12.4
354 317˘30.7 ´10.4 10.0 6.8
0.6 0.6˘0.1 ´0.1 16.8 13.9
2.2 2.2˘0.3 ´4.3 14.0 10.3
Worenine
55.5 51.3˘6.1 ´7.4 12.0 12.0
1776 1715˘90.6 ´3.4 4.9 7.6
1.1 1.1˘1.1 0.8 14.3 9.2
4.4 5.3˘0.6 14.1 11.6 8.4
Berberrubine
111 112˘14.0 1.5 13.1 6.2
3536 3949˘437 11.7 11.3 9.4
0.2 0.2˘0.03 13.4 13.8 1.8
0.8 0.8˘0.1 2.1 12.7 9.9
Coptisine
20.0 22.4˘2.6 11.8 11.9 9.0
640 555˘70.3 ´13.3 13.1 8.4
Molecules2016,21,913 8of15
2.2.4. ExtractionRecoveryandMatrixEffects
The mean recovery and matrix effect data were evaluated by analyzing QC samples at three
concentrations levels (LQC, MQC, and HQC) with six replicates. The data on the extraction
recoveryandthematrixeffectsofeightalkaloidsdeterminedintheplasmaaredepictedinTable5.
MeanrecoveriesofeightanalytesoftheQCsampleswereintherangeof86.4%–100.0%,andthemean
recoveryoftheI.S.was88.7%˘13.7%. ThematrixeffectsderivedfromtheQCsamplesrangedfrom
85.2%to106.8%,andfortheI.S.,itwas98.2%˘11.1%. Thus,itwasconfirmedthatLLEwithacetone
wasafeasibleandappropriatemethodfortheextractionoftheanalytesandtheI.S.,andmoreover,the
methodwasreliableandlittlematrixeffectoccurred.
Table5.Matrixeffectsandextractionrecoveryfortheanalytesinratplasma(n=6).
SpikedConcentration MatrixEffect ExtractionRecovery
Compounds
(ng/mL)
Mean(%) RSD(%) Mean(%) RSD(%)
2.0 105.1 8.4 94.8 14.2
Tetrahydropalmatine 50.7 100.5 8.2 95.9 7.8
1622 104.4 7.2 94.7 4.2
0.4 101.7 6.2 93.1 13.6
Palmatine 10.7 93.7 11.2 86.4 5.1
342 101.8 12.5 95.6 3.0
4.3 99.1 11.4 93.4 5.5
Magnoflorine 108 85.2 13.1 90.0 4.1
3456 98.5 13.4 86.4 14.3
2.2 100.0 13.5 100.0 6.2
Columbamine 55.8 98.8 5.9 90.8 8.9
1784 101.2 9.6 98.4 12.0
0.4 101.8 12.3 92.3 8.2
Berberine 11.1 100.5 9.3 93.2 4.0
354 102.3 5.0 97.6 11.1
2.2 92.5 7.8 98.9 12.1
Worenine 55.5 87.7 10.0 88.0 9.8
1776 105.6 5.1 93.4 1.8
4.4 94.7 2.4 90.6 10.2
Berberrubine 111 98.0 11.3 91.2 2.6
3536 100.8 4.3 93.4 11.5
0.8 106.8 3.9 92.3 7.4
Coptisine 20.0 99.5 5.7 92.2 4.2
640 89.1 5.1 89.0 8.4
I.S. 2000 98.2 11.1 88.7 13.7
2.2.5. Stability
Freeze-thawstability,short-termstability,long-termstabilityandpost-preparativestabilitywere
determinedbymeasuringtheresponsearearatio(analyte/I.S.)ofstabilitysamplesagainstfreshly
prepared QC samples with identical concentrations. Summary of the stability data of the eight
analytesunderdifferentconditionsareshowninTable6. Themeasuredconcentrationsfortheeight
alkaloidsateachQClevelsdeviatedwithin14.9%,whichindicatedthatthemethodisapplicablefor
routineanalysis.
Molecules2016,21,913 9of15
Table6.Stabilitiesoftheanalytesinratplasma(n=6).
Stability(%RE)
Compounds SpikedConcentration
(ng/mL) Freeze-Thaw ShortTerm LongTerm PostPreparative
2.0 ´10.9 13.3 13.6 2.8
Tetrahydropalmatine 50.7 ´13.8 12.5 13.5 ´0.8
1622 6.2 8.0 14.5 ´5.5
0.4 ´0.2 14.9 5.9 8.5
Palmatine 10.7 ´2.3 ´9.6 ´11.2 ´12.4
342 ´2.8 ´1.7 12.9 ´3.0
4.3 12.2 10.6 9.4 10.7
Magnoflorine 108 ´3.4 14.1 13.6 ´1.0
3456 13.3 7.4 13.2 6.7
2.2 12.3 11.9 11.7 6.9
Columbamine 55.8 ´10.2 ´0.3 ´2.5 ´11.2
1784 1.9 ´4.2 ´5.9 ´1.8
0.4 9.4 1.2 2.4 2.9
Berberine 11.1 ´12.5 ´1.0 ´14.6 2.6
354 ´12.5 ´4.1 ´11.0 ´4.2
2.2 5.6 ´12.9 ´7.8 12.3
Worenine 55.5 4.7 8.9 9.9 5.6
1776 ´5.4 ´9.8 ´4.3 ´6.6
4.4 8.1 8.2 8.0 13.9
Berberrubine 111 11.3 0.01 ´2.5 4.4
3536 6.9 8.6 ´0.6 11.2
0.8 13.1 ´14.4 8.7 5.1
Coptisine 20.0 2.1 ´6.1 ´5.6 1.8
640 8.4 ´4.8 0.6 12.2
2.3. MethodComparisonwithExistingReports
Thereareseveralreportsonassayingberberine,coptisine,palmatineinRhizomeCoptidis[28–30],
but no analytical method was developed for the simultaneous determination of the given eight
alkaloidsinthisstudy. Moststudiesmainlyfocusedonthedeterminationofseveralkindsofalkaloids
presentinhighcontents. Therewasareportonthesimultaneousdeterminationofcoptisine,berberine,
palmatine,magnoflorineandcolumbaminefromwine-processedRhizomaCoptidisinratplasma[8].
TheLLOQswere0.5ng/mLforcoptisineandcolumbamine,2.03ng/mLforberberine,2.2ng/mLfor
palmatine,and5.5ng/mLformagnoflorine. TheLLOQinthisstudywas0.1ng/mLforpalmatine
and berberine, 0.2 ng/mL for coptisine, 0.5 ng/mL for columbamine, tetrahydropalmatine and
worenine and 1.1 ng/mL for berberrubine and magnoflorine. The chromatographic run time was
9 min. In comparison, the method described in this study is much shorter and more suitable for
clinicalapplications. RhizomaCoptidisisamainherbintraditionalChinesemedicineprescriptions
suchasXilixiaocapsule,ShensongyangxincapsuleandYiqingCapsulewhichhasseveralspecies
andexplicitboundaries. Toimprovetheunderstandingofthedifferencebetweenpharmacokinetics
and even pharmacology of these crude herbs, a selective and sensitive analytical method for the
simultaneousquantificationofthemainbioactivecomponentsinthisherbafteroraladministrationof
TCMinplasmaisrequired. Tothebestofourknowledge,thisisthefirsttimetetrahydropalmatine,
palmatine,magnoflorine,columbamine,berberine,worenine,berberrubineandcoptisinehasbeen
simultaneously determined using an LC-MS-MS method. Furthermore, Figure 3 shows the mean
plasma concentration-time profiles of the eight alkaloids: tetrahydropalmatine (A); palmatine (B);
magnoflorine(C);columbamine(D);berberine(E);worenine(F);berberrubine(G);coptisine(H)inrat
plasmaafteroraladministrationofCCYandcf. Theinformationdescribedabovemightbehelpful
forfurtherstudiesonthepharmacokineticsofCCYandCFandbeneficialforapplicationofthese
TraditionalChineseMedicinesinclinicaltherapy. Moreover,ithasthepotentialtoprovidevaluable
contributionstothefieldofplanttoxinanalysesfortoxicitystudies.
Molecules 2016, 21, 913 9 of 14
4.3 12.2 10.6 9.4 10.7
Magnoflorine 108 −3.4 14.1 13.6 −1.0
3456 13.3 7.4 13.2 6.7
2.2 12.3 11.9 11.7 6.9
Columbamine 55.8 −10.2 −0.3 −2.5 −11.2
1784 1.9 −4.2 −5.9 −1.8
0.4 9.4 1.2 2.4 2.9
Berberine 11.1 −12.5 −1.0 −14.6 2.6
354 −12.5 −4.1 −11.0 −4.2
2.2 5.6 −12.9 −7.8 12.3
Worenine 55.5 4.7 8.9 9.9 5.6
1776 −5.4 −9.8 −4.3 −6.6
4.4 8.1 8.2 8.0 13.9
Berberrubine 111 11.3 0.01 −2.5 4.4
3536 6.9 8.6 −0.6 11.2
0.8 13.1 −14.4 8.7 5.1
Coptisine 20.0 2.1 −6.1 −5.6 1.8
640 8.4 −4.8 0.6 12.2
2.3. Method Comparison with Existing Reports
There are several reports on assaying berberine, coptisine, palmatine in Rhizome Coptidis [28–30],
but no analytical method was developed for the simultaneous determination of the given eight
alkaloids in this study. Most studies mainly focused on the determination of several kinds of alkaloids
present in high contents. There was a report on the simultaneous determination of coptisine, berberine,
palmatine, magnoflorine and columbamine from wine-processed Rhizoma Coptidis in rat plasma [8].
The LLOQs were 0.5 ng/mL for coptisine and columbamine, 2.03 ng/mL for berberine, 2.2 ng/mL for
palmatine, and 5.5 ng/mL for magnoflorine. The LLOQ in this study was 0.1 ng/mL for palmatine and
berberine, 0.2 ng/mL for coptisine, 0.5 ng/mL for columbamine, tetrahydropalmatine and worenine and
1.1 ng/mL for berberrubine and magnoflorine. The chromatographic run time was 9 min. In comparison,
the method described in this study is much shorter and more suitable for clinical applications.
Rhizoma Coptidis is a main herb in traditional Chinese medicine prescriptions such as Xilixiao capsule,
Shensong yangxin capsule and Yiqing Capsule which has several species and explicit boundaries. To
improve the understanding of the difference between pharmacokinetics and even pharmacology of
these crude herbs, a selective and sensitive analytical method for the simultaneous quantification of
the main bioactive components in this herb after oral administration of TCM in plasma is required.
To the best of our knowledge, this is the first time tetrahydropalmatine, palmatine, magnoflorine,
columbamine, berberine, worenine, berberrubine and coptisine has been simultaneously determined
using an LC-MS-MS method. Furthermore, Figure 3 shows the mean plasma concentration-time
profiles of the eight alkaloids: tetrahydropalmatine (A); palmatine (B); magnoflorine (C); columbamine
(D); berberine (E); worenine (F); berberrubine (G); coptisine (H) in rat plasma after oral administration
of CCY and cf. The information described above might be helpful for further studies on the
pharmacokinetics of CCY and CF and beneficial for application of these Traditional Chinese
Molecules2016,21,913 10of15
Medicines in clinical therapy. Moreover, it has the potential to provide valuable contributions to the
field of plant toxin analyses for toxicity studies.
A Tetrahydropalmatine B Palmatine
70.0 80.0
L)60.0 70 Coptis deltoidea C. Y. cheng et Hsiao L) 70.0 7800 Coptis deltoidea C.Y.cheng et Hsiao
Concentration(ng/m1234500000.....00000 12345600000000 1Coptis2 chinens3is Franc4h 5 6 Concentration(ng/m 123456000000......000000 12345600000000 1 Copt2is chinen3sis Fran4ch 5 6
0.0 0.0
Molecules0 2016, 214, 913 8 12 16 20 24 0 4 8 12 16 20 10 o24f 14
time(h) time(h)
C Magnoflorine D Columbamine
mL)345000..00 334050000 CCooppttiiss dchelintoeindseias FCr.Yan. cchheng et Hsiao mL)110200..00 110200 CCooppttiiss dcheiltnoeindseias FCr.Yan.cchheng et Hsiao
Concentration(ng/112230505000000.....00000 1122505050000000 1 2 3 4 5 6 Concentration(ng/468000...000 246800000
20.0 0 1 2 3 4 5 6
50.0
0.0 0.0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
time(h) time(h)
E Berberine F Worenine
120.0 120 Coptis deltoidea C.Y. cheng et Hsiao 50.0 50 Coptis deltoidea C.Y.cheng et Hsiao
mL) 100.0 100 Coptis chinensis Franch mL)4405..00 4405 Coptis chinensis Franch
ration(ng/ 6800..00 468000 ntration(ng/22330505....0000 112233050505
cent 40.0 20 once15.0 05
on 20.0 0 C 10.0 0 1 2 3 4 5 6
C 0 1 2 3 4 5 6 5.0
0.0 0.0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
time(h) time(h)
G Berberrubine Coptisine
H
60.0
120.0 120
60 Coptis deltoidea C.Y.cheng et Hsiao
ncentration(ng/mL)23450000....0000 12345000000 CCooppttiiss dchelintoeindseias FCr.Ya.n cchheng et Hsiao Concentration(ng/mL) 146800000....0000 1246800000000 1 Copti2s chinen3sis Fran4ch 5 6
Co10.0 0 1 2 3 4 5 6 20.0
0.0 0.0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
time(h) time(h)
FFiigguurree 33.. MMeeaann ccoonncceennttrraattiioonn--ttiimmee pprrooffiilleess ooff tteettrraahhyyddrrooppaallmmaattiinnee ((AA));; ppaallmmaattiinnee ((BB));; mmaaggnnoofflloorriinnee ((CC));;
ccoolluummbbaammiinnee ((DD));; bbeerrbbeerriinnee ((EE));; wwoorreenniinnee ((FF));; bbeerrbbeerrrruubbiinnee ((GG));; ccooppttiissiinnee ((HH)) iinn rraatt ppllaassmmaa aafftteerr oorraall
aaddmmiinniissttrraattiioonn ooff CCCCYY aanndd ccff.. EEaacchh ppooiinntt rreepprreesseennttss tthhee mmeeaann ˘± SSDD ((nn == 1144)). .
3. Experimental Section
3. ExperimentalSection
33..11.. MMaatteerriiaall aanndd RReeggeennttss
TThhee eeiigghhtt rreeffeerreennccee ssttaannddaarrddss ((AA––HH,, ppuurriittyy 9988..00%%)) wweerree aallll oobbttaaiinneedd ffrroomm tthhee CChheennggdduu PPuuffeeii DDee
BBiiootteecchh CCoo..,, LLttdd.. ((CChheennggdduu,, SSiicchhuuaann,, CChhiinnaa)).. TThhee II..SS.. ((bbiiffeennddaattee)) wwiitthh aa ppuurriittyy ooff 9999..99%% wwaass ppuurrcchhaasseedd
ffrroomm tthhee NNaattiioonnaall IInnssttiittuutteess ffoorr FFoooodd aanndd DDrruugg CCoonnttrrooll ((BBeeiijjiinngg,, CChhiinnaa)).. WWaatteerr ((HH22OO)) wwaass ppuurriiffiieedd
bbyy aa MMiillllii--QQ ssyysstteemm ((MMiilllliippoorree,, BBiilllleerriiccaa,, MMAA,, UUSSAA)) iinn oouurr llaabboorraattoorryy.. HHPPLLCC--ggrraaddee mmeetthhaannooll aanndd
acetonitrile were purchased from Amethyst Chemicals (Beijing, China). HPLC-grade acetic acid was
purchased from CNW Technologies (Düsseldorf, Germany). All other reagents were analytical-grade.
CF was the dried rhizome of the Coptis chinensis Franch which was purchased from Bozhou
Medicinal Material Company (Anhui, China) and authenticated by Professor Zhenyue Wang in
Heilongjiang University of Chinese Medicine. CCY was the dried rhizome of the Coptis deltoidea C. Y.
cheng et Hsiao which was collected from Hongya County, Meishan City, Sichuan Province, China, which
is protected as a vulnerable plant in China [31], and also authenticated by Professor Zhenyue Wang.
3.2. Preparation of CCY Extract and CF Extract
CCY and CF were crushed to pieces before use. A dried powder sample of each (100 g) was
extracted three times (1 h each time) under reflux with 70% ethanol at a material:liquid ratio of 1:10
and finally the extracted solution was concentrated. The residue was reconstituted in water to obtain