利用质谱及高效液相法分析极性脂质

关于利用质谱分析及高效液相分析磷脂混合物中的极性磷脂的文档

JournalofChromatographyA,1279 (2013) 98–107

ContentslistsavailableatSciVerseScienceDirect

JournalofChromatographyA

journalhomepage:http://wendang.chazidian.com/locate/chrom

a

Simultaneouspro?lingofpolarlipidsbysupercritical?uid

chromatography/tandemmassspectrometrywithmethylation?

JaeWonLeea,ShinNishiumib,MasaruYoshidab,c,d,EiichiroFukusakia,TakeshiBambaa,?

a

DepartmentofBiotechnology,GraduateSchoolofEngineering,OsakaUniversity,2-1Yamadaoka,Suita,Osaka,565-0871,Japan

DivisionofGastroenterology,DepartmentofInternalMedicine,KobeUniversityGraduateSchoolofMedicine,7-5-1Kusunoki-cho,Chu-o-ku,Kobe,Hyogo,650-0017,Japanc

TheIntegratedCenterforMassSpectrometry,KobeUniversityGraduateSchoolofMedicine,7-5-1Kusunoki-cho,Chu-o-ku,Kobe,Hyogo,650-0017,Japand

DivisionofMetabolomicsResearch,DepartmentofInternalMedicine,KobeUniversityGraduateSchoolofMedicine,7-5-1Kusunoki-cho,Chu-o-ku,Kobe,Hyogo,650-0017,Japan

b

article

info

abstract

Articlehistory:

Received3November2012Receivedinrevisedform22December2012

Accepted3January2013

Available online 10 January 2013

Keywords:MethylationPolarlipid

Supercritical?uidchromatographyTandemmassspectrometry

Supercritical?uidchromatography/tandemmassspectrometry(SFC/MS/MS)withmethylationwasusedforthesimultaneouspro?lingofdiversepolarlipidsinamixture.Ahighthroughput,highresolutionanal-ysisofnineteenclassesofpolarlipidsincludingphospholipids,lysophospholipids,andsphingolipidswasperformedin6min.Methylationbytrimethylsilyl-diazomethanesuppressedpeaktailingandimproveddetectionsensitivityofphosphatidylserine(PS),phosphatidicacid(PA),lysophosphatidylserine(LPS),lysophosphatidylinositol(LPI),lysophosphatidicacid(LPA),ceramide-1-phosphate(Cer1P),sphingosine-1-phosphate(So1P),andsphinganine-1-phosphate(Sa1P).ThelimitsofdetectionforPS,PA,LPS,LPI,LPA,Cer1P,So1P,andSa1Pwereenhanced7.5-,26.7-,600-,116.7-,500-,75-,3000-,and4500-fold,respec-tively.Globalqualitativeandquantitativeanalysisofnotonlythehigh-abundancespeciesbutalsothelow-abundancespeciesinthepolarlipidswasachieved.Whenthemethodwasappliedtomouseliver,4PSs,24PAs,3lysophosphatidylethanolamines,11LPSs,6lysophosphatidylglycerols,4LPIs,13LPAs,7sphingomyelins,11Cer1Ps,So1P,andSa1Pwereadditionallyanalyzed.Furthermore,thequanti?cationofvariousmolecularspeciesineachpolarlipidwascarriedout.

© 2013 Elsevier B.V. All rights reserved.

1.Introduction

Lipidsplaycrucialrolesinenergystorage,cellmembranecom-ponents,cellularsignaling,andcell–cellinteractionsintissues,cellmembranes,andorganelles[1].Manystudieshaveprovidedvalu-ableinsightsintotherelationshipbetweenmetabolicchangesinlipidsanddiseases/disorders[2–8].Furthermore,alteredlipidpro-?lesinbiologicalspecieshavebeenscreenedtoidentifybiomarkers[9,10].Therefore,therehasbeenincreasedinterestinlipidomicstoobtainalipidpro?leandtogainacomprehensiveunderstandingofthefunctionsoflipidsinabiologicalsample[11–14].

Phospholipids(PLs),lysophospholipids(LPLs),andsphin-golipids(SLs)areclassi?edaspolarlipids.PLsaremajorcomponentsofthecellularmembrane.LPLsfunctionasligandsfornumeroussignalingreceptors.PLsandLPLshavedifferenttypesofheadgroupsandcombinationsoffattyacidsthatvaryinchainlengthanddegreeofsaturation[15].SLshaverolesincellular

?ThestudyrepresentsaportionofthedissertationsubmittedbyJaeWonLeetoOsakaUniversityinpartialful?llmentoftherequirementforhisPh.D.

?Correspondingauthor.Tel.:+810668797418;fax:+810668797418.E-mailaddresses:bamba@bio.eng.osaka-u.ac.jp,tbamba@tim.hi-ho.ne.jp(T.Bamba).

membranecomponentsaswellasbioactivecompoundshav-ingcrucialbiologicalfunctions.Anyvariationintheheadgroupattachedtotheprimaryhydroxylgroup,N-acylgroup,andsphingoid-basebackbonewouldcauseachangeinthenatureandcharacteristicsoftheSL[16].Therefore,comprehensivepro?lingofpolarlipidsinamixtureremainsamajorchallenge.

Severalchromatographicmethodssuchashighperformanceliquidchromatography(HPLC)[17,18],ultra-highperformanceliquidchromatography(UHPLC)[19,20],andsupercritical?uidchromatography(SFC)[21,22]arewidelyusedfortheseparationofpolarlipids.Furthermore,high-sensitivity,high-selectivitymassspectrometry(MS)[23,24]iseffectivefortheidenti?cationandquanti?cationofvariousclassesoflipids.Detailedpro?lingofPLshasbeenperformedbyreversed-phase(RP)HPLC[25].However,thesemethodsarenotwellsuitedforthesimultaneousanaly-sisofphosphatidicacid(PA)andphosphatidylserine(PS)http://wendang.chazidian.comedastartingmobilephasecon-tainingalowconcentrationofphosphoricacid(5?mol/L)andahighconcentrationofwater(40%)[26]anddevelopedRP-LCcon-ditionstoreducePAandPSpeaktailing.However,peaktailingforlysophosphatidicacid(LPA)couldnotbecompletelyelimi-nated,andlow-abundancePLscouldnotbeanalyzed.Moreover,theuseofhighamountsofphosphoricacidcangiveharmfuleffectsontheESIprobe.Satoetal.foundthatprewashingthe

0021-9673/$–seefrontmatter© 2013 Elsevier B.V. All rights reserved.http://wendang.chazidian.com/10.1016/j.chroma.2013.01.020

关于利用质谱分析及高效液相分析磷脂混合物中的极性磷脂的文档

J.W.Leeetal./J.Chromatogr.A1279 (2013) 98–107

99

analyticalcolumnusingethylenediaminetetraaceticacid(EDTA)inPLanalysisresultedinimprovedpeakshapeandsensitivitytoPA,PS,andLPA[27].Further,quantitativepro?lingoflow-abundancePLsinbiologicalsampleswaspossible.Leeetal.successfullyappliednano?owLC/MS/MStothesimultaneousandglobalpro-?lingof62PLsand50LPLsinhumanplasma[28],buttheycouldnotanalyzethelow-abundancePS,PA,andlysophosphatidylser-ine(LPS).Ag-adductionhasalsobeenusedfortheregiochemicalstudyofPLs[29].Inaddition,LC/MS/MShasemergedasapow-erfultoolfortheanalysisofSLs[30,31].Amethodforquantifyingthecellularlevelsofphospho-SLssuchasceramide-1-phosphate(Cer1P)andsphingosine-1-phosphate(So1P)hasbeendeveloped[32];inparticular,improvedSo1Panalysisbyderivatizationwithnaphthalene-2,3-dicarboxaldehydehasbeendemonstrated[33].

However,thereisnoreportedmethodforthesimultaneouspro?lingofPLs,LPLs,andSLs.TheeffectivenessofSFC/MSinana-lyzingalipidmixture—amixtureof14typesofdiverselipids,includingglycolipids,neutrallipids,PLs,andSLs—waspreviouslydemonstrated[21],buttheanalysisofpolarlipidswasnotef?-cientbecauseofseverepeaktailingandlowsensitivity.Further,theapplicationofEDTAwasnotclari?edtoimprovetheSLanal-ysis[27].Forthecomprehensiveanalysisofpolarlipids,anewapproachthatallowsfortheef?cientanalysisofphospho-SLsaswellasPSandPAisnecessary.Inrecentdecades,varioustypesofderivatizationhavebeenappliedtoimprovethereso-lutionandpeakshapeinHPLCandtoenhancetheselectivityandionizationef?ciencyinMS[34].Therefore,inourpreviousstudy,weappliedtrimethylsilyl(TMS)derivatizationforpolarlipidpro?lingbySFC/MS/MS[35]andfoundthatderivatizationimprovedthepeakshapeandsensitivitytoPA,phosphatidylin-ositol(PI),LPA,lysophosphatidylinositol(LPI),andSo1P.However,http://wendang.chazidian.comedtrimethylsilyldiazomethane(TMSD)toquantifyphosphatidylinositol(3,4,5)-trisphosphate(PtdIns(3,4,5)P3)incellsandtissues[36].TheuseofTMSDallowedforrapidandcompletemethylationofthefreehydroxylgroupsinthephosphatemoi-etiesandthusenhancedtheef?ciencyofPtdIns(3,4,5)P3analysis.Methylationcouldalsobeusedfortheanalysisofcompoundswithphosphategroups.

Variouspolarlipidssuchasphosphatidylcholine(PC),phos-phatidylethanolamine(PE),PS,phosphatidylglycerol(PG),PI,PA,lysophosphatidylcholine(LPC),lysophosphatidylethanolamine(LPE),LPS,lysophosphatidylglycerol(LPG),LPI,LPA,sphingomyelin(SM),Cer1P,So1P,andsphinganine-1-phosphate(Sa1P)containphosphategroups.Therefore,weappliedTMSDmethylationtoanalyzesixPLs,sixLPLs,andfourSLs.Herein,wedescribeahigh-resolution,high-throughputmethodbasedonSFC/MS/MSwithmethylationforthecomprehensivepro?lingofsixteenmethylatedpolarlipidsandthreenonmethylatedSLssuchasceramide(Cer),sphingosine(So),andsphinganine(Sa).Furthermore,wereportthesensitiveandreliablequanti?cationoflow-abundancepolarlipidsinmouseliver.

2.Materialsandmethods

2.1.Chemicals

Carbondioxide(99.9%grade;NerikiGas,Osaka,Japan)wasusedasthemobilephase.HPLC-grademethanol(KishidaChemical,Osaka,Japan)containing0.1%(w/w)ammoniumformate(99.99%;Sigma–Aldrich,Milwaukee,WI,USA)wasusedasthemodi?er.Detailsofthepolarlipidstandards(AvantiPolarLipids,Inc.)areasfollows:PC(12:0–12:0),PC(12:0–13:0),PE(12:0–13:0),PS(12:0–13:0),PG(12:0–13:0),PI(12:0–13:0),PA(12:0–13:0),LPC(C17:0),LPE(C14:0),LPS(C16:0),LPG(C14:0),LPI(C18:1),LPA

(C14:0),SM(d18:1–17:0),Cer(d18:1–17:0),Cer1P(d18:1–12:0),Cer1P(d18:1–16:0),So(d17:1),So1P(d17:1),Sa(d17:0),andSa1P(d17:0).TMSDwaspurchasedfromTokyoChemicalIndustryCo.,Ltd.(Tokyo,Japan).

2.2.Samplepreparation

Methanolsolutionsofthepolarlipidstandards(1?mol/L)wereprepared,storedat?30?C,andthendilutedtothedesiredconcen-tration.MaleC57BL/6Jmiceweresacri?cedunderanesthesia,andthentheliverswereobtainedquickly.Theperfusedliverswerepre-paredwithnormalsaline,andwerekeptat?80?Cbeforeuse.TenmgofmouseliverimmersedinliquidN2washomogenizedinaballmillmixerMM301(Retsch,Haan,Germany)for1minat20Hz.Thehomogenatewasspikedwith1000?Lofinternalstandard(IS)solu-tion(0.1?mol/LofPC(12:0–13:0),PE(12:0–13:0),PS(12:0–13:0),PG(12:0–13:0),PI(12:0–13:0),PA(12:0–13:0),LPC(17:0),SM(d18:0–17:0),Cer(d18:1–17:0),Cer1P(d18:1–12:0),So(d17:1),So1P(d17:1),Sa(d17:0),andSa1P(d17:0)inmethanol).Aftervortexingandincubatingonicefor10min,themixturewascen-trifuged(10,000×g,5min,4?C).Finally,900?Lofthesupernatantwasusedforpolarlipidpro?ling.

2.3.TMSDmethylation

TMSDshowsacutetoxicityforinhalation,causingcentralner-voussystemdepression,drowsiness,dizziness,andlungdamage.Althoughthisreagentisnotexplosivelikediazomethane,extremecareshouldbetakenwhenhandingit.Forthisreason,methylationbyTMSDhasbeencarriedoutinafumehood,withtheuseofade-quatepersonalsafetyequipment.AsolutionofTMSD(2mol/L)inhexane(50?L)wasaddedtothelipidextracts(100?L)fromthemouseliverandthelipidstandardsamples(300?L)dissolvedinmethanoltoobtainyellow-coloredsolutions.Aftervortexingfor30s,methylationwasperformedat50?Cfor10min(optimizedconditions).Additionofglacialaceticacid(6?L)quenchedthemethylationandaffordedcolorlesssamples,whichwerethensub-jectedtoSFC/MS/MSanalysis.

2.4.SFC/MS/MSanalysis

SFC/MS/MSanalysiswasperformedbyusingAnalyticalSFCMethodStation(Waters,Milford,MA,USA),whichincludeda?uiddeliverymodule,anAliasautosampler,anAnalytical-2-Prepcolumnoven,a2998photodiodearraydetector,a3100massdetector,anautomatedbackpressureregulator(ABPR),andaXevoTQ(Waters,Milford,MA,USA)massspectrometer.TheSFCandtriplequadruple(QqQ)MSsystemswerecontrolledbySuperChromautomatedcontrolmodulesoftwareandMassLynxsoftware,respectively.The?owrateofthemobilephasecontain-ingthemodi?er,backpressure,andcolumntemperatureweresetto3mL/min,10MPa,and37?C,respectively.Foreachrun,5?Lofthesamplewasinjectedbythefullsampleloopinjectionmethod.Fortheanalysisofmethylatedpolarlipids,thefollowingmodi?ergradientconditionswereemployed:startingmodi?er,20%(v/v);rampingto25%(v/v)over5min;returnto20%(v/v)over1min;totalruntime,6min.Ontheotherhand,thegradientconditionsfortheanalysisofnonmethylatedpolarlipidswereasfollows:start-ingmodi?er,20%(v/v);rampingto25%(v/v)over5min;holdingfor5min;returnto20%(v/v)over1min;totalruntime,11min.QqQMSanalysiswasperformedinthepositiveionmodeofelec-trosprayionization(ESI),underthefollowingconditions:capillaryvoltage,3000V;sourcetemperature,150?C;desolvationtemper-ature,350?C;conegas?owrate,50L/h;desolvationgas?owrate,

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100J.W.Leeetal./J.Chromatogr.A1279 (2013) 98–107

Table1

OptimizedMRMconditionsofpolarlipidpro?lingbynonmethylationandmethylation.

Polarlipids

Ionmode

MRMtransitions

CV(V)

MS/MSCE(eV)

NonmethylationPCPositive[M+H]>184

2930PEPositive

[M+H]>[M+H-141]2320PS

Positive[M+H]>[M+H-185]

2519PG

Positive[M+NH4]>[M+NH4-189]1516PI

Positive[M+H]>[M+H-260]

2217PAPositive[M+NH4]>[M+NH4-115]1816LPCPositive

[M+H]>184

3126LPE

Positive[M+H]>[M+H-141]2018LPS

Positive[M+H]>[M+H-185]2221LPGNegative

[M?H]>[M?H-228]3725LPI

Positive[M+Na]>2834030LPANegative[M?H]>1532822SMPositive[M+H]>1843026Cer1PPositive

[M+H]>2642030So1P

Positive[M+H]>2642015Sa1PPositive[M+H]>2662420CerPositive

[M+H]>264

1928So

Positive[M+H]>[M+H-18]1615Sa

Positive[M+H]>[M+H-18]

25

15MethylationPCPositive[M+H]>198

4026PEPositive[M+H]>[M+H-155]2720PSPositive[M+H]>[M+H-213]2622PGPositive

[M+H]>[M+H-186]2418PI

Positive[M+H]>[M+H-274]2726PA

Positive[M+H]>[M+H-126]2218LPC

Positive[M+H]>198

3827LPEPositive[M+H]>[M+H-155]2318LPSPositive[M+H]>[M+H-213]2421LPGPositive[M+H]>[M+H-186]1916LPIPositive

[M+Na]>297

4028LPA

Positive[M+H]>[M+H-126]1716SM

Positive[M+H]>1983629Cer1P

Positive[M+H]>2642233So1PPositive[M+H]>2642215Sa1PPositive

[M+H]>266

22

20

800L/h;collisiongas?owrate,12mL/h;MScollisionenergy(CE),20V;extractorvoltage,3V.

3.Resultsanddiscussion

3.1.TMSDmethylationforpolarlipidpro?ling

TMSDwasusedformethylationofthefreehydroxylgroupsinthephosphatemoietiesofpolarlipids.Thenumberofadductedmethoxygroupsdifferedwiththetypeoflipids:PC,1;PE,1;PS,2;PG,1;PI,1;PA,2;LPC,1;LPE,1;LPS,2;LPG,1;LPI,1;LPA,2;SM,1;Cer1P,2;So1P,2;Sa1P,2.Cer,So,andSa,whichhadnophosphategroup,werenotmethylated(Fig.1).Thisisthe?rstattempttouseTMSDmethylationforpolarlipidpro?lingbySFC/MS/MS.There-fore,eachlipidstandardwasusedtodeveloptheMS/MSmethodforthepro?lingofmethylatedpolarlipids.InMS/MS,themulti-plereactionmonitoring(MRM)wasusedtomonitoraparticularfragmentionofaselectedprecursorion[37].TheparametersofMRM,whichiscommonlyusedinMS/MS,includenumberofMRMtransitions(precursorm/z>fragmentm/z),conevoltage(CV),andMS/MSCE.Byelectrosprayionization(ESI),?fteenmethylatedandthreenonmethylatedpolarlipidsweredetectedas[M+H]+ions,andmethylatedLPIwasdetectedas[M+Na]+ioninthepositiveionmode.

Byproductionscan,aprecursorionandfragmentionsweredetectedintheMS/MSspectrum(Fig.2),andthefragmentionwiththehighestintensitywasselectedfortheMRMtransition(Table1).Theneutrallossduringcollision-induceddissociation(CID)dif-feredfordifferentpolarlipids:PEandLPE,155Da(C3H11NO4P);

Table2

Methylationef?ciencyofsixteenpolarlipidsinamixture.

Polarlipids

Thepercentageofmethylatedcompounds(%)a

0Meb

1Me

2Me

PC67.7±4.4c32.3±4.4–PE0.5±0.499.5±0.4–

PS0.9±0.10.7±0.198.4±0.2PG6.8±0.193.2±0.1–PI7±0.593±0.5–

PA0.2±0.23±0.696.8±0.8LPC62.1±7.737.9±7.7–LPE0.9±0.199.1±0.1–

LPS2.9±0.51.4±0.195.7±0.6LPG7±0.293±0.2–LPI1.1±0.398.9±0.3–

LPA0.2±0.124.9±5.874.9±5.9SM67.1±5.832.9±5.8–

Cer1P1.1±0.15.1±0.193.8±0.2So1P19±0.52.3±0.578.7±1Sa1P

11.5

±

0.1

7.3

±

0.4

81.2±0.5

a

(Peakareaofacompoundinanonmethylatedmixture?peakareaofanon-methylatedcompoundinamethylatedmixture/peakareaofacompoundinanonmethylatedmixture)×100(%).b

Thenumberofadductedmethoxygroup.c

Thevaluesofpercentagesaremean±SD(n=3).

PSandLPS,213Da(C5H12NO6P);PGandLPG,186Da(C4H11O6P);PAandLPA,126Da(C2H7O4P);andPI,274Da(C7H15O9P).Meth-ylatedphosphorylcholine(m/z198)wasdetectedasthefragmentionintheanalysisofPC,LPC,andSM,whilemethylatedinositolmonophosphateadductedbyNa+(m/z297)wasthefragmentioninLPIanalysis.ThefragmentionintheanalysisofCer,Cer1P,andSo1Pwasthesphingoidbase.Forexample,inthecaseofd17:1andd18:1,thefragmentionswerem/z250andm/z264respec-tively.ThefragmentionofSa1P,too,wasthesphingoidbase:d17:0,m/z252andd18:0,m/z266.TheneutrallossofSoandSawas18Da(H2O).Finally,eachMRMtransitionforapolarlipidwasopti-mized.Subsequently,theCVandMS/MSCEvalueswereoptimizedbycomparingthepeakintensitiesandsignal-to-noiseratios(S/Ns)obtainedfromtheprogrammedconevoltages,whichrangedfrom15to45V.

Next,thetemperature(tested:10,20,30,40,50,and60?C)andtime(tested:5,10,15,and20min)forTMSDmethylationwereoptimized,andeachpolarlipidstandardwasmethylatedthreetimes(n=3)underthetestedconditions.PC(12:0–12:0)wasusedastheinternalstandard,andtherelativepeakarea(com-pound/internalstandard)wasexamined.Slightdifferenceswereobservedunderthedifferentsetsofconditionsemployed,and?nally,methylationwasoptimizedat50?Cfor10min(Supple-mentalFig.1).Inaddition,themethylationef?ciencyofeachpolarlipidinamixturewascharacterizedtocon?rmwhethermeth-ylationwasapplicableforthepolarlipidpro?lingofabiologicalsample.Thenumberofadductedmethoxygroupsdiffered(oneortwo)accordingtothemolecularstructureofthelipids.There-fore,therelativepeakareaforlipidswithtwo,one,ornomethoxygroupsinamethylatedmixturehadtobeexamined.Methylatedandnonmethylatedcompoundsmusthavedifferentionizationef?-ciencyaccordingtothemolecularstructures,sothatitisinsuf?cienttocomparesimplythepeakareaofthem.Therefore,weappliedthepeakareasofanonmethylatedcompoundinbothmethylatedandnonmethylatedmixturestotestthemethylationef?ciencyinamixture.Finally,themethylationef?ciencyofsixteenpolarlipidsinamixture,i.e.,thepercentageofmethylatedcompounds((peakareaofacompoundinanonmethylatedmixture?peakareaofanonmethylatedcompoundinamethylatedmixture)/peakareaofacompoundinanonmethylatedmixture×100(%)),wasexamined(Table2).Mostpolarlipidswereeffectivelymeth-ylated(>74.9%).However,choline-containingpolarlipidswere

关于利用质谱分析及高效液相分析磷脂混合物中的极性磷脂的文档

J.W.Leeetal./J.Chromatogr.A1279 (2013) 98–107

101

Fig.1.Molecularstructureofmethylatedpolarlipids.Theadductedmethoxygroupsweremarkedwithanemptybox.

methylatedwithlowef?ciency:PC,32.3%;LPC,37.9%;andSM,32.9%.Furthermore,inordertotesttherepeatabilityofmethyla-tion,eachpolarlipidwasmethylatedsixtimes(n=6)undertheoptimizedconditions.Thevariationsintherelativepeakareaswereexpressedintermsoftherelativestandarddeviation(RSD(%)):PC,7.5%;PE,3.1%;PS,3.3%;PG,7.9%;PI,6.4%;PA,5.7%;LPC,7.7%;LPE,5.1%;LPS,4.0%;LPG,6.2%;LPI,7.4%;LPA,6.9%;SM,6.4%;Cer1P,7.6%;So1P,6.7%;andSa1P,6.6%.AlltheRSDswerelessthan8%,indicatingthehighrepeatabilityofmethylationforpolarlipidpro?ling.

Supplementarydatarelatedtothisarticlefound,intheonlineversion,athttp://wendang.chazidian.com/10.1016/j.chroma.2013.01.020.

3.2.Simultaneouspro?lingofpolarlipidsbySFC/MS/MS

Toidentifythemosteffectivecolumnforpolarlipidpro?lingbySFC/MS/MS,ODS,C8,diol,andcyano(CN)columnsweretestedbyexaminingtheresolutionandpeakshapeofsixPLstandardsineach

case;inparticular,peakshapewasconsideredthemostimportantcriterionfordecidingtheoptimumcolumn.Polarlipidsweresepa-ratedeffectivelyontheYMCPack-CNcolumn(250mm×4.6mmID;5?m,Waters),butmethylatedPCwasnotdetected.YMCPack-Diolcolumn(250mm×4.6mmID;5?m,Waters)wasinef-fectiveforthedetectionofmethylatedPC,andpeaktailingwasobservedformethylatedPE.SeveralODScolumns,too,gavepeaktailingforPC.TheInertsilODS-EPcolumn(250mm×4.6mmID;5?m,GLSciences)gavehigherresolutionthandidtheothercolumnsbutresultedinpeaktailingformethylatedPE.Inert-silODS-4(250mm×4.6mmID;5?m,GLSciences),InertSustainC18(250mm×4.6mmID;5?m,GLSciences),andInertsilC8-4(250mm×4.6mmID;5?m,GLSciences)columnsofferedgoodpeakshapesforsixPLs,althoughtheresolutionobservedwaspoor(SupplementalFig.2).Thus,theInertsilODS-4columnwaschosenforpolarlipidpro?lingwithmethylation.

Supplementarydatarelatedtothisarticlefound,intheonlineversion,athttp://wendang.chazidian.com/10.1016/j.chroma.2013.01.020

.

Fig.2.MS/MSspectrumdataofmethylatedPS(12:0–13:0,m/z666)byproductionscan(MS/MSCE,15eV).

关于利用质谱分析及高效液相分析磷脂混合物中的极性磷脂的文档

102J.W.Leeetal./J.Chromatogr.A1279 (2013) 98–107

Fig.3.Multiplereactionmonitoring(MRM)dataofthestandardPC(12:0–13:0),PE(12:0–13:0),PS(12:0–13:0),PG(12:0–13:0),PI(12:0–13:0),PA(12:0–13:0),LPC(C17:0),LPE(C14:0),LPS(C16:0),LPG(C14:0),LPI(C18:1),LPA(C14:0),SM(d18:1–17:0),Cer1P(d18:1–16:0),So1P(d17:1),andSa1P(d17:0)by(A)nonmethylationand(B)methylation.

Next,themodi?erratio,whichaffectsthepeakshapeandretentiontime(RT),wasoptimizedafterexaminingthreemodi-?ergradients:10–15%(v/v),15–20%(v/v),and20–25%(v/v).PolarlipidstandardswereanalyzedbyusingtheInertsilODS-4columnwithdifferentgradientmodi?ersfor6min.Whenthegradientwas10–15%(v/v),poorpeakshapesandlowpeakintensitieswereobtained,whileagradientof15–20%(v/v)gavegoodresultsonly

forPC,PI,andPA.Hence,themodi?ergradientthatgavethehighestpeakintensityforthepolarlipids,20–25%(v/v),waschosen.

Theresultsobtainedforsimultaneouspolarlipidpro?lingwithandwithoutmethylationwerecomparedtoprovetheeffectivenessofthedevelopedSFC/MS/MSmethod.Sixteenpolarlipidstan-dards,includingPC,PE,PS,PG,PI,PA,LPC,LPE,LPS,LPG,LPI,LPA,SM,Cer1P,So1P,andSa1P,wereusedforthecomparison.Inthe

Table3

ValidationofmethylatedpolarlipidanalysisandtheLODscomparisonwithnonmethylatedlipidanalysis.

Polarlipids

RTa(min)

Intradayvariationb

Interdayvariationc

Correlation(R2)

Linearrange(fmol)

LOD(fmol)

RTd

PCPEPSPGPIPALPCLPELPSLPGLPILPASMCer1PSo1PSa1PCerSoSa

Peakareae

RTd1.11.21.00.91.10.51.21.00.71.01.40.80.70.60.90.70.40.60.9

Peakareae

Methylated

Nonmethylated0.252150252502004.53030005017,5001250

1.515075022501052.5

1.641.71.741.691.681.871.481.41.461.361.491.412.112.11.381.392.861.661.69

1.01.21.10.61.20.31.01.20.61.11.30.70.80.70.60.60.40.61.1

2.04.15.25.67.92.85.29.39.03.99.02.86.11.59.45.44.67.46.0

7.19.76.58.58.84.86.29.47.54.510.44.87.76.19.85.55.88.16.2

0.99530.99560.99980.99310.99240.99900.99110.99810.99240.99110.99720.99800.99260.99250.99310.99800.99420.99240.9922

2.5–500050–500020–50005–5000200–50,0007.5–25001–250015–50005–50007.5–5000150–25,0002.5–50009–25002–50000.25–50000.5–500010–50005–50002.5–5000

2.5502052007.511557.51502.5920.250.5–––

abcde

Retentiontime.

RSD(n=6)(%)ofintradayvariation.

RSD(n=18)(%)ofinterdayvariation(3days).

Relativeretentiontime(compound/internalstandard).Relativepeakarea(compound/internalstandard).