VincentDesfontainea,1,GioacchinoLucaLosaccoa,1,YoricGagnebina,JulianPezzattia, WilliamP.Farrellb,VíctorGonzález-Ruiza,SergeRudaza,Jean-LucVeutheya,
DavyGuillarmea,∗
aSchoolofPharmaceuticalSciences,UniversityofGeneva,UniversityofLausanne,CMU–RueMichelServet1,1211Geneva4,Switzerland
bPfizer,Inc.,WorldwideMedicinalChemistry,LaJollaLaboratories,10770ScienceCenterDrive,SanDiego,CA92121,USA
a r t i c le i n f o
Articlehistory:
Received4April2018
Receivedinrevisedform20May2018 Accepted26May2018
TheaimofthisstudywastoevaluatethesuitabilityofSFC-MSfortheanalysisofawiderangeof com-poundsincludinglipophilicandhighlyhydrophilicsubstances(logPvaluescomprisedbetween−6and 11),foritspotentialapplicationtowardhumanmetabolomics.Forthispurpose,agenericunified chro-matographygradientfrom2to100%organicmodifierinCO2wassystematicallyapplied.Intermsof chemistry,thebeststationaryphasesforthisapplicationwerefoundtobetheAgilentPoroshellHILIC (baresilica)andMacherey-NagelNucleoshellHILIC(silicabondedwithazwitterionicligand).Toavoid systemoverpressureatveryhighorganicmodifierproportion,columnsof100×3mmI.D.packedwith sub-3!msuperficiallyporousparticleswereselected.Intermsoforganicmodifier,amixtureof95%
MeOHand5%waterwasselected,with50mMammoniumformateand1mMammoniumfluoride,to affordgoodsolubilityofanalytesinthemobilephase,limitedretentionforthemosthydrophilic metabo-litesandsuitablepeakshapesofionizablespecies.Asamplediluentcontaining50%ACN/50%waterwas employedasinjectionsolvent.
These conditionswere applied toa representativeset ofmetabolitesbelonging to nucleosides, nucleotides,smallorganicacids,smallbases,sulfated/sulfonatedmetabolites,poly-alcohols,lipidrelated substances,quaternaryammoniummetabolites,phosphate-basedsubstances,carbohydratesandamino acids.Amongallthesemetabolites,65%ofthecompoundswereadequatelyanalyzedwithexcellentpeak shape,23%provideddistortedpeakshapes,whileonly12%werenotdetected(mostlymetaboliteshaving severalphosphateorseveralcarboxylicacidgroups).
©2018ElsevierB.V.Allrightsreserved.
1. Introduction
Metabolomicsisdefinedasthesystematicidentificationand quantificationofsmallmoleculesknownasmetabolites,produced by the metabolism of living organisms, in different biological fluids[1].Itgenerally requirestheuseofhighlypowerful ana-lytical techniques, such as nuclear magnetic resonance (NMR) spectroscopyormassspectrometry(MS),thelatterusually
cou-∗Correspondingauthor.
E-mailaddress:davy.guillarme@unige.ch(D.Guillarme).
1Theseauthorscontributedequally.
pled to different chromatographic techniques [1]. The interest onmetabolomicshasrapidlyincreasedamongscientistssinceit represents avaluableapproach toperform clinicaldiagnosis in precisionmedicine[2,3].Thereare,however,severalissueslinked tometabolomicanalysis.Inparticular,itimpliestheanalysisof anextremelywiderangeofmolecules,possessingquitediverse physico-chemicalproperties[1,3].Currently,thestate-of-the-art approachconsistsincombiningreversedphaseliquid chromatog-raphy(RPLC)andhydrophilicinteractionchromatography(HILIC) withhigh-resolutionMSinstruments,suchas quadrupole-time-of-flightorquadrupole-OrbitrapMS[4].Thesetwochromatographic approachespresentsomechallenges.Indeed,RPLCisnotidealto analyzethemostpolarmetabolites,duetothelowretentionofsuch https://doi.org/10.1016/j.chroma.2018.05.055
0021-9673/©2018ElsevierB.V.Allrightsreserved.
V.Desfontaineetal./J.Chromatogr.A1562(2018)96–107 97 analytesonclassicalRPLCstationaryphases(C4,C8orC18)[5,6].In
HILICmode,polarmetabolitescanbesufficientlyretained[7,8],but thetechniqueisnotadaptedtothemostlipophilicsubstances,and remainssometimesdifficulttouse(i.e.lackofrepeatability, sig-nificantimpactofsamplediluent,complexandmultipleretention mechanisms,etc.)[9,10].
Inthiscontext,supercriticalfluidchromatography(SFC), cou-pled to MS, haspotentialasan interestingalternativeto RPLC andHILIC.Initially,SFCwasdevelopedasasubstitutetonormal phaseliquidchromatography(NPLC)[11],fortheanalysisofhighly hydrophobicsubstances,suchaslipidsorpetrochemicalsamples, duetothenon-polarcharacteristicsofCO2.SFCwas,also,used insteadofliquidchromatographyforcompoundsof pharmaceu-ticalinterestinbothacademia[12]andindustry[13,14],aswellas inotherprocessessuchasimpuritycontrol[15]Morerecently,SFC hasbeenemployedfortheanalysisofsubstanceswithincreasing polarity[16–21],thankstotheadditionofpolarorganicmodifier andsaltsinthemobilephase.Forinstance,Taguchietal.applied SFCforthesimultaneousanalysisofliposolubleandhydrosoluble vitamins[22].Intheirwork,theauthorsdemonstratedthat com-poundswithlogPvaluesbetween−2.1and10.1canbeanalyzed inthesamerunwitha genericSFCgradientvaryingfrompure CO2topureMeOH.Thesuggesteddesignationforsucha gradi-entwasunifiedchromatography(UC),asitwasabletomakethe linkbetweensupercriticalconditionsatthebeginningofthe gradi-entandliquidchromatographyattheend.Moreover,SFCisknown tobefullycompatiblewithMS,oftenresultinginimproved sensi-tivitycomparedtoRPLC,thankstothepresenceofsolventswith alowsurfacetensioninthemobilephase(i.e.methanol)andthe absence(orlimitedpresence)ofwater[23–25].Italsopossesses adifferentbehaviortowardsmatrixeffectsthanLC–MS,showing predominantlyionsuppressionwhile,inLC–MS,ionenhancement ismorecommon[25–27].
TheaimofthisstudywastoevaluatetheapplicabilityofSFC-MS tothefieldofmetabolomicsandtofindoutsomegenericSFC condi-tionsapplicabletoatrainingsetof57representativemetabolites, coveringabroadrangeoflogP,from−6to11.Forthispurpose, a comparisonof variouscolumnchemistries aswellasmobile phaseconditions(organicsolventchoiceandcomposition, addi-tivesnatureandconcentration,temperature,etc.)wascarriedout.
Anevaluationofthekineticperformanceandthebehaviorofthe SFC-MSinterfaceathighpercentageofco-solventwasalsostudied.
2. Materialandmethods
2.1. Chemicals,reagentsandcolumns
All metabolites, reported in Table S1 of the supplementary material,wereobtainedfromSigma-Aldrich(Buchs,Switzerland).
Thesemetaboliteswereselectedbasedonthehumanmetabolome database(HMDB)[28],whichcontainsabout100,000entries rep-resentingavariedselectionofhumanmetabolites belongingto various chemical classes. In the present work, 57 representa-tivemetaboliteswereselected,basedontheiravailability,price, diversityandrepresentabilityofalltheHMDBchemicalclasses.
Thechemicalclassescoveredbytheselectedmetabolitesinclude sulfated/sulfonated metabolites, nucleosides, nucleotides, small organicacids,smallbases,poly-alcohols,lipidrelatedsubstances, quaternaryammoniummetabolites,phosphate-basedsubstances, carbohydratesandaminoacids.
Methanol(MeOH),acetonitrile(ACN),isopropanolofOPTIMA LC/MS grade and water ofUHPLC grade were purchased from FisherScientific(Loughborough,UK).Ammoniumformate(AmF), ammonium acetate andammonium fluoride (NH4F)were pur-chased from Sigma-Aldrich. Pressurized carbon dioxide (CO2)
3.0grade (99.9%) was purchased from PanGas (Dagmerstellen, Switzerland). Sevendifferent columns were compared, namely Poroshell HILIC 2.7!m (Agilent, Santa Clara, CA, USA), Nucle-oshellHILIC2.7!m(Macherey-Nagel,Düren,Germany),Sunshell Diol 2.6!m and Sunshell 2-EP 2.6!m (ChromaNik Technolo-giesINC.,Osaka,Japan),Cosmosil3-hydrophenyl2.5!m(Nacalai TesqueINC.,Kyoto,Japan),KinetexC18Polar2.6!mandSynergi Polar2.5!m(Phenomenex,Torrance,CA,USA).Forsakeof com-parison,allthe selectedcolumnswereofthesamedimensions (100×3.0mm).
2.2. UHPSFC-MS/MSinstrumentation
AllexperimentswereperformedonaWatersAcquityUPC2 sys-tem(Waters,Milford,MA,USA)equippedwithaBinarySolvent Managerdeliverypump, aSampleManagerautosamplerwhich includeda10!Lloopforpartialloopinjection,acolumnovenand atwo-step(activeandpassive)backpressureregulator(BPR). Ace-tonitrileandamixtureofMeOH/H2O50/50wereusedastheweak andstrongwashsolvents,respectively,withvolumesof600!Land 200!L.ThechromatographicsystemwashyphenatedtoaWaters TQDtriplequadrupoleviaadouble-TsplitterinterfacefromWaters.
ThehyphenationinterfaceandsplitterforUHPSFC-MS/MSwere describedelsewhere[29].Additionalmake-upsolventforSFC-MS operationwasbroughttothesystembyaWatersIsocraticSolvent Manager(ISM)pump,deliveringpureMeOHat0.3mL/min.
TheTQDdetectorwasoperatedinbothpositiveandnegative electrosprayionization(ESI)modesandthedifferentparameters wereoptimizedtoobtainthehighestsensitivity:source tempera-tureat150◦C,desolvationtemperatureat450◦C,capillaryvoltage at3.0kV.Nitrogenwasusedasadesolvationgasat600L/hr,while argonwasusedasacollisiongasat0.10mL/min.Otherparameters suchasconevoltagesandcollisionenergiesweretuned, depend-ingontheanalyte,inarangeof10–40Vand5–20eV,respectively, asdescribedinTableS1.Dwelltimesweresetupbetween8and 20ms,dependingonthesamples,tohaveasufficientnumberof datapointsacrosseachchromatographicpeak.MassLynx4.1 soft-warewasusedforinstrumentcontrolanddataacquisition.
2.3. Samplepreparation
Samplestocksolutionswerepreparedbydissolvingtheselected compoundsinasuitablesamplediluent,accordingtotheir physico-chemicalproperties.AsreportedinTableS1,eitherwater,0.1N NaOHin water,0.1N HClinwater,MeOH,ethanol (EtOH), iso-propanol(IPA),tetrahydrofuran(THF),orchloroform(CHCl3)were used,tohave allthestock solutionsata finalconcentrationof 1mg/mL.Stocksolutionswerethenstoredat−22◦C.Priorto injec-tion,stocksolutionswerethawedandsamplesweredilutedtoa concentrationof20!g/mL,byadding980!Lofthesolventusedin thestocksolutionsto20!Lofsample.
Sixmixtureswereprepared(TableS2),witheachmoleculeata suitableconcentration,tohavecomparablesignaltonoiseratios.
Themixturesweredilutedtoafinalvolumeof1mL.
2.4. Chromatographicconditions
Theinitialcompositionofthemobilephasewas98%CO2/2%
organicmodifier,andwasheld constantfor1min,witha sub-sequent5-minlineargradientuptoafinalcompositionof100%
organicmodifier.Afteranisocraticstepof1minat100%organic modifier,thecolumnwasreconditionedwithinitialconditionsfor 1.5min.Back-pressurewasmaintainedconstantat120bar,while mobilephasetemperaturewaskeptat40◦C.Allcompoundswere
98 V.Desfontaineetal./J.Chromatogr.A1562(2018)96–107 injectedonallcolumnswithaninjectionvolumeof1!L,either
individuallyorasmixtures.
3. Resultsanddiscussion
3.1. KineticevaluationofSFCwithunifiedchromatography gradient
Recently,thepopularityofSFChasstronglyincreasedthanks tothecommercializationofmoderninstrumentation,compatible with columns packed with sub-2!m particles, and a signifi-cantnumberofrecentSFCapplicationshavebeenreportedwith suchcolumns.However,thegeneratedbackpressureremainshigh [15,30–32],evenwithsupercriticalorsubcriticalfluids,whilethe upperpressurelimitsofUHPSFCsystemsaremorelimitedthan thoseofUHPLC.Forthisreason,theamountofco-solventinthe mobilephasehastobekeptinareasonablerange,tomaintain opti-malkineticperformance.AtypicalUHPSFCgradientrangesfrom 2to40%co-solvent,whichisadequatetoelutehighlylipophilic andmoderatelypolarcompounds.However,SFChasalsoproven itsapplicabilitytotheanalysisofmorepolarcompounds,anda new trendappeared,withan increaseofthepercentage of co-solvent upto100% duringthegradient.Under suchconditions, the use of columnspacked with sub-2!m particles cannot be envisioned.Indeed,theelevatedbackpressuregeneratedbyhigh co-solventpercentageinthemobilephasewouldurgeSFCusers toapplyrelativelylowflowrates.Asanexample,the experimen-talbackpressuresobservedwithdifferentmobilephaseconditions whenusinga100×3.0mmcolumnpackedwith1.7!mparticles werereportedinFig.1A.OnourUHPSFCsystem(WatersAcquity UPC2),theuppersystempressure limit(approximately400bar) wasreachedat2.8and2.0mL/minwithpureCO2and25%MeOHin CO2,respectively.Theseflowratesarefullyacceptableforageneric UHPSFCmethod,takingintoaccountcolumndimensions.However, whenthepercentageofMeOHwasfurtherincreased,theflowrate shouldbegreatlyreducedtomaintainareasonablebackpressure.
Asanexample,whenusingamobilephasecomposedof75%MeOH inCO2,theflowratewaslimitedto0.8mL/min.Oneattractive solu-tiontoreducethepressureinvolvestheuseofcolumnspackedwith superficiallyporous particles(SPP),alsoknown ascore-shellor fused-coretechnology.Thankstotheirparticularshape,these par-ticles,withadiameterofc.a.2.6!m,areabletoreachcomparable efficienciestofullyporoussub-2!mparticles,butatlower back-pressure.Theexperimentalbackpressuresobservedwiththesame mobilephasecompositionsasinFig.1A,whenusinga100×3.0mm columnpacked with2.6!mSPP,werereportedinFig. 1B. The differencewasobvious,andaflowrateof1.4mL/mincouldnow bereached,evenwithpureMeOHasmobilephase.Inconclusion, theuseofsuchcolumnsrepresentsaviableandinteresting solu-tionwhenunifiedgradientsfrom2to100%co-solventhavetobe appliedinSFC.
SFChasalwaysbeenrecognizedforits excellentkinetic per-formance [33]. Indeed, due to the reduction of mobile phase viscosityandimprovementofdiffusioncoefficientscomparedto liquidconditions,bothoptimallinearvelocityandmasstransferare drasticallyenhancedcomparedtoRPLC[34].Thisistruein super-criticalorsubcriticalconditions,butthekineticperformancewith higherproportionofco-solventhastobeevaluated,sinceithasnot beenyetreportedintheliterature.Forthispurpose,fourdifferent metaboliteswereanalyzedunderisocraticconditionsatdifferent co-solventpercentagesonthesamecolumn,namelyNucleoshell HILIC.Onthiscolumn,aretentionfactorof5wasobtainedwhen analyzingcaffeine,melatonine,maleicacidandindoxylsulphateat 2,18,40and80%ofco-solventinCO2,respectively.The correspond-ingVanDeemtercurveswerethenconstructedforeachcompound
(seeFig.2).InpureSFCconditions(caseofcaffeine),theshapeofthe curvewasexpected,witharelativelyhighoptimallinearvelocity of7–8mm/s,andaflatcurveduetoalimitedcontributionofthe masstransferresistance.Insubcriticalconditions(caseof mela-toninandmaleicacid),theVanDeemtercurveshapeswerequite different.Indeed,the mobilephase viscosity andmasstransfer resistancewereincreasedduetothehigherproportionoforganic modifierinthemobilephase,andconsequently,theoptimal lin-earvelocitydecreasedto2–3mm/s.Thiswasevenmorenoticeable forindoxylsulphate,whichwaselutedwith80%co-solventinCO2 wheretheconditionscouldbeconsideredascomparabletoRPLC andtheresistancetomasstransferwasthereforegreatlyincreased.
Theoptimallinearvelocitywasalsodrasticallyreducedwithan optimalvaluearound1mm/s(correspondingtoaflowrateofonly 0.3mL/minona3.0mminternaldiametercolumn).Ifweconsider thesimultaneous analysis ofthesefourmetabolites,a gradient rangingfromaverylowpercentage(2%)toahighproportionof co-solvent(possibly100%)hastobeused.However,aspreviously discussed,theoptimallinearvelocity(orflowrate)isnotthesame forallthesemetabolites,whichraisesthequestionoftheidealflow rateinthisparticularsituation.Ontheonehand,anelevatedlinear velocityisnotadaptedtothehighlyretainedcompoundseluted withhighorganicmodifierproportionandwouldgeneratesystem overpressure.However,alowlinearvelocityisnotfavorablefor weaklyretainedcompounds(lowkineticperformance,according tothevanDeemtercurves)andwouldincreaseanalysistime.In consequence,thebestcompromisewastoselectanintermediate linearvelocitybetween3and4mm/s,correspondingtoaflowrate between0.8and1mL/minona3.0mmI.D.column.Another inter-estingapproachwouldhavebeentoimplementadecreasingflow rategradient(simultaneouslywiththeorganicmodifiergradient).
Inthisway,ahighflowratecouldbeusedatthebeginningofthe gradient,toeluteweaklyretainedmetabolites.Then,theflowrate wouldbeprogressivelydecreased,inordertofitwiththelower optimallinearvelocityrequirementobservedathighco-solvent percentage,andtomaintainaconstantbackpressureduetothe increasingmobilephaseviscositytoavoidsystemoverpressure.
Thisapproachwasnotselectedinordertokeepgenericconditions easilyimplementedandtoavoidirreproducibleretentiontimes.
3.2. BehaviouroftheSFC-MSinterfaceathighco-solvent percentages
Thenatureandspecificityoftheavailableinterfacesfor hyphen-ating SFC and MS have been substantially studied [29,35,36].
Nowadays,themostprevalentandpromisinginterfacefor electro-sprayionization(ESI)operationseemstobethe“pre-BPRsplitter withsheathpump”interface[36]usedinthisstudy.However,the behaviorofthisinterfacewasonlydescribedforgenericSFC con-ditionswithproportionsofco-solventlowerthan50%.Themain featuresoftheinterfacethatneedtobeevaluatedarei)theamount ofMeOHreachingtheionizationsource,ii)thesplitratiobetween theflowgoingtothesourceandtothewaste,andiii)thedilution factorinducedbytheadditionalmake-upsolventintheinterface.
Thecalculationsforamobilephasecontaininganimportant frac-tionofco-solventweremadeandresultsweregraphicallyreported inFigs.S1andS2ofthesupplementarymaterial.Formore infor-mationaboutthecalculations,thereaderisreferredto[36].
Theflow-rateoftheorganicmodifierreachingtheESIsource withagivenSFC-MSsetupisanimportantparameter.Ifitistoo high,theconditionsmaynotbeidealforthesprayformationin theESIsource.Ontheotherhand,analytesprecipitationcanoccur inthetubingfromtheinterfacetotheMSsourceifthisflow is toolow,duetoCO2decompressioncooling(endothermicreaction).
Generally,aflowrateofapproximately250!L/minisconsidered toavoidbothissues.Atareasonableco-solventpercentageof20%
V.Desfontaineetal./J.Chromatogr.A1562(2018)96–107 99 Fig. 1.Total backpressure as a func-tion of the SFC pump flow rate for an Acquity UPC2 Torus 2-PIC 1.7!m, 100×3.0mm column (A)and a Nucle-oshellHILIC2.7!m,100×3.0mmcolumn (B).Differentmobilephaseconditionsare represented:pureCO2(blue),CO2/MeOH 75:25 (red), CO2/MeOH 50:50 (green), CO2/MeOH25:75(purple)andpureMeOH (orange).Mobile phase temperature of 40◦Cand backpressureof150bar.(For interpretationofthereferencestocolour inthisfigurelegend,thereaderisreferred tothewebversionofthisarticle.)
organicmodifier,theflowenteringtheESIsourceishighly depen-dentonthemobilephaseandmake-upsolventflowrates,andvary between120and370!L/min,asreportedinFig.S1A.Therefore, themake-upflowratehastobecarefullyselectedbasedonthe SFCpumpflowrate.Thistrendisreducedwhenthepercentage ofco-solventisincreasedto40%(see Fig.S1B).Inthese condi-tions,theflowrateenteringtheESIsourcewascomprisedbetween 180and330!L/minandthemake-upflowratewasmuchless critical.Finally,withahigherco-solventpercentage(seeFig.S1C, correspondingto80%),theflow-rateoftheorganicmodifier enter-ingtheESIsourcewascompletelyleveledataround270!L/min (forMeOH).Undertheseconditions,themake-upsolventaddition couldbestoppedtoreducesolventconsumption.Nevertheless,the make-upsolvent couldstillbemaintainedifitsnaturestrongly differsfromtheoneoftheco-solvent,andifitcouldprovidea sensitivityimprovement[37].Finally,inthecaseofunified chro-matographygradient,thebestsolutionwouldbetoimplementa decreasingsolventaddition,toimprovetheionizationofthe early-elutingmetabolitesandreducesolventconsumptionduringthe elutionofthestronglyretainedmetabolites.Inthepresentwork, thelatterapproachwasnotselected,sinceitcouldhaveraisedsome issuesregardingthenormalizationoftheMSsignalsgivenbythe compounds[38].Amake-upflowrateof0.3mL/minofMeOHwas finallyselectedasthebestcompromiseforunifiedchromatography gradient.
ThedilutionfactorduetotheSFC-MSgeometry(relevantfor concentrationdependentdetector,suchasESI/MS)wasreported inFig.S2foramobilephasecontaining80%MeOHinCO2.Itisdue totheadditionofthemake-upsolvent,andonlydependsontheSFC
ThedilutionfactorduetotheSFC-MSgeometry(relevantfor concentrationdependentdetector,suchasESI/MS)wasreported inFig.S2foramobilephasecontaining80%MeOHinCO2.Itisdue totheadditionofthemake-upsolvent,andonlydependsontheSFC