determination of N,N-dimethyltryptamine in rat plasma and brain;

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ContentslistsavailableatScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rn al h om e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a

Heart-cutting two-dimensional liquid chromatography coupled to quadrupole-orbitrap high resolution mass spectrometry for

determination of N,N-dimethyltryptamine in rat plasma and brain;

Method development and application

Tímea Körmöczi

^a,b

, Írisz Szabó

^c

, Eszter Farkas

^c

, Botond Penke

^b

, Tamás Janáky

^b

, István Ilisz

^a

, Róbert Berkecz

^a,b,∗

aInstituteofPharmaceuticalAnalysis,FacultyofPharmacy,UniversityofSzeged,SomogyiUtca4,H-6720,Szeged,Hungary

bDepartmentofMedicalChemistry,FacultyofMedicine,UniversityofSzeged,Dómtér8,H-6720,Szeged,Hungary

cDepartmentofMedicalPhysicsandInformatics,FacultyofMedicine,UniversityofSzeged,Korányifasor9,H-6720,Szeged,Hungary

a r t i c l e i n f o

Articlehistory:

Received21June2020

Receivedinrevisedform30August2020 Accepted2September2020

Availableonline9September2020

Keywords:

N,N-dimethyltryptamine

␣-methyltryptamine Ratplasma Ratbrain

Heart-cutting2D-LC-HRMS/MS

a b s t r a c t

Theorthogonalheart-cuttingliquidchromatography(LC)modescoupledtohigh-resolutiontandemmass spectrometry(HRMS/MS)provideanumberofpossibilitiestoenhanceselectivityandsensitivityforthe determinationoftargetedcompoundsincomplexbiologicalmatricies.Herewereportthedevelopmentof anewfast2D-LC-(HRMS/MS)methodanditssuccessfulapplicationforquantitativedeterminationofthe levelofplasmaandbrainN,N-dimethyltriptamine(DMT)using␣-methyltryptamine(AMT)asinternal standardinanexperimentalmodelofcerebralischemia/reperfusionusingDMTadministration.The2D- LCseparationwascarriedoutbyacombinationofhydrophilicinteractionliquidchromatography(HILIC) inthefirstdimensionfollowedbysecond-dimensionalreversed-phase(RP)chromatographywithina totalruntimeof10min.TheenrichmentofHILICeffluentofinterestcontainingDMTwasperformed usingaC18trappingcolumn.Duringmethoddevelopmentseveralparametersofsamplepreparation procedures,chromatographicseparationandmassspectrometricdetectionwereoptimisedtoachieve highDMTrecovery(plasma:90%,brain:88%)andsensitivity(plasma:0.108ng/mLofLOD,brain:0.212 ng/gofLOD)applyingtargetedanalyticalmethodwithstrictLCandHRMSMSconfirmatorycriteria.

Concerningratplasmasample,theconcentrationofDMTbeforehypoxia(49.3–114.3ng/mLplasma) wasgenerallyhigherthanthatafterhypoxia(10.6–96.1ng/mLplasma).Aftertreatment,theconcentra- tionofDMTinbrainwaselevateduptotherangeof2–6.1ng/g.

Overall,ouranalyticalapproachissuitabletodetectandconﬁrmthepresenceofDMTadministeredto experimentalanimalswiththerapeuticpurposeinareliablemanner.

1. Introduction

DMTis an endogenous hallucinogen and traceamine found invarioustissues suchasthebrain,pineal glandandlung, and bodyﬂuidssuchasurine,cerebrospinalﬂuidandbloodplasmain mammals[1–4].DMTisproducedbysomaticcellsunderphysi- ologicalconditions[1,3],anditsconcentrationhasbeenfoundto increaseinresponsetopathophysiologicalhomeostaticchallenges (e.g.hypoxiaoroxidativestress)[1,3].Concurrently,DMTisbest

∗Correspondingauthor.

E-mailaddress:berkecz.robert@pharm.u-szeged.hu(R.Berkecz).

knownasanindolealkaloidtypicallysynthesised byplants like PsychotriaviridisandDiplopteryscabrerana[5].

AlthoughDMTwastracedwithgas-chromatographyinbody ﬂuidsofpsychiatricpatientsandcontrolsubjects[3,4],itsphysi- ologicalroleisstilltobeunderstood,inparticular,becauseDMT undergoesrapidenzymaticdegradationbymonoamineoxidases (MAO)[3].Ontheotherhand,DMThasbeenimplicatedinneuro- protectionathighconcentration,eithersynthesisedinthenervous tissueinresponsetohypoxiaoroxidativestress,ororiginatingfrom thelungand reachingthebrainviathebloodstream[1,6].The putativeneuroprotectiveactionofDMThasbeenutilisedinexper- imentalmodelsofcerebralorrenalischemia.DMTadministeredat supraphysiologicalconcentrationtoexperimentalrodentsresulted inasmallercerebralinfarctsize[7]andpromotedcellsurvivalina

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cellcultureofhumancerebrocorticalneuronsexposedtohypoxia [8].

EarlyresearchesofDMTfocusedprimarilyonthepsychological andpharmacologicaleffectsofconsumingayahuasca[5].During the last 60 years, many studies reported finding DMT and its metabolitesin animal tissueand humanurine,blood and cere- brospinalfluidusingliquidchromatography(LC).Nowadaysliquid chromatography–tandemmassspectrometry(LC–MS/MS)isone ofthemostwidelyusedbioanalyticalmethodsforquantificationof smallmoleculesinbiologicalsamples.Itisduetoitshighsensitivity andhighselectivity,thusbeingabletoprovidemorereliabledata thantheLCtechniqueitself,whichisalsotrueoftheanalysisofDMT [9],onlyafewstudiesreportedthepresenceofDMTinmammalian tissue;forinstance,inpinealglandmicrodialysateofrat[2],ratkid- ney,lung,liver,brain,humanlungandrabbitliver[10].According tocertainviewpoints,detectionofendogenousDMTisnotrele- vantwhenit isexogenouslyadministereddue tothedissimilar concentrationrange[11].

The ultimatepurpose of ourupcoming study is toevaluate theneuroprotectiveactionofDMTinanexperimentalmodelof cerebralischemia/reperfusion,aggravatedbytransientanoxiaand recurrentspreadingdepolarisations.Here,wesetouttoestablish anovel,highlysensitiveanalyticaltooltodeterminetheconcen- tration of endogenous DMT in our model and to measure the accumulationof intravenouslyadministered exogenousDMT in bloodplasmaandbraintissue.

In modern pharmaceutical analysis, quantitation of analyte or analytes of interest in complex biological matrices requires highselectivityand sensitivity.Nowadays,two-dimensionalliq- uidchromatography(2D-LC)isoneofthemostpowerfulanalytical toolstoimprovethequalityofobtaineddata,especiallyincom- bination withHRMS/MS. The comprehensiveand heart-cutting analysesareknowntobethemaintechniquesof2D-LC.Theheart- cuttingmodeisatargeted2Dtechnique,becauseonlyaselected eluentfraction(singleheart-cutting)orfractions(multipleheart- cutting)arecollected,trappedforsecond-dimensionalseparation [12–14].

Nowadays, the typical methodutilised for determination of administeredDMTinplasmaandbrainsampleisreversedphase chromatographicseparationcoupledtotandemmassspectrome- tryusingelectrosprayoratmosphericpressurechemicalionization inpositivemode[15–18].However,thehighresolutionaccurate HRMS/MSmethod,incombinationwithorthogonal2D-LCsepara- tionincomparisonwithonedimensionalLC-HRMS/MSmethods, hastheabilitytoprovidemorereliabledata.Furthermore,itgives theopportunitytouseloweramountsofsampleswithsimilaror betterlimitof detectionfor DMT.The requiredamountof bio- logicalsample isa critical issue,especiallyin pharmacokinetics assaysorwhentheavailablesampleis limitedforinstanceata repeatedsamplingofbloodfromratandmouse.Anotherkeyfac- torofmethoddevelopmentisruntime,whichshouldbekeptas lowaspossible.FordeterminationofDMTincomplexbiological samplessuchasbrainandplasma,theapplicationofthegradient elutionisessential,thereforethetotalruntimeofanalysisincreases bythetimeofcolumnwashandequilibration.Intheliterature,the totalruntimeofgradientLC–MS/MSanalysisofDMTcanbefound intherangeof11.5−25mininbrainandplasmasamples[15–18].

Thus,themaingoalsofthisstudywerethedevelopmentofafast heart-cutting2D-LC-HRMS/MSmethodandoptimisationofsam- plepreparationproceduresforthequantitativeanalysisofDMTin ratbrainandplasmausingarelativelylowamountofbiological samples.Themethodthusdevelopedwasappliedforthedeter- minationofDMTlevel inratplasmaand brainofexperimental model of cerebral ischemia/reperfusionusing DMT administration.

2. Materialsandmethods 2.1. Chemicalsandstandards

N,N-Dimethyltryptamine (DMT) standard with purity >98.5

% was obtain from Lipomed AG (Arlesheim, Switzerland). ␣- Methyltryptamine(AMT)withpurity>99%waskindlyprovided bytheDepartmentofForensicMedicine,FacultyofMedicine,Uni- versity of Szeged, Hungary, and used as internal standard (IS).

Acetonitrile,ammoniumformate,formicacid,methanol,water,(all LC–MSgrade),35%ammoniasolution,ethylacetate(HPLCgrade) and reagentgrade perchloric acid(70 %) werepurchased from VWR(Radnor,PA,USA).Isoﬂurane(MedicusPartnerLtd.,Biator- bágy,Hungary)forinvivopreparations,theanaestheticisoﬂurane wasproducedbyCP-Pharma(HandelsgesellschaftmbH,Germany);

atropineandlidocainewerepurchasedfromEgisPharmaceuticals Ltd.(Budapest,Hungary);EDTAandpargilinfromSigma-Aldrich, (StLouis,MO, USA);chloralhydrate fromMolarChemicals Ltd.

(Budapest,Hungary),andsterilephysiologicalsalinefromB.Braun AG,(Melsungen,Germany).

2.2. Animalsandtissuesamples

TheexperimentalprocedureswereapprovedbytheNational Food Chain Safety and Animal Health Directorate of Csongrád County,Hungary(licenseNo.XXXII./2015).Theprocedureswere performedaccordingtotheguidelinesoftheScientiﬁcCommittee ofAnimalExperimentationoftheHungarianAcademyofSciences (updatedLawandRegulationsonAnimalProtection:40/2013.(II.

14.)Gov.ofHungary),followingtheEUDirective2010/63/EUon theprotection.

Young3monthsold,maleSprague-Dawleyrats(CharlesRiver Laboratories,328±18g)wereusedinthisstudy.Standardrodent chowandtapwaterweresuppliedadlibitum.Theanimalswere housedunderconstanttemperature,humidityandlightingcondi- tions(23^◦C,12:12hlight/darkcycle,lightsonat7a.m.).

Wesetouttodeterminethebloodplasmaandbrainconcentra- tionofDMTsynthesisedbytissuesinresponsetoischemia/hypoxia (i.e.endogenousDMT),andDMTcontentofbloodplasmaandbrain tissueafterDMTadministeredtotheanimalsbyintravenous(i.v.) infusion(i.e.exogenousDMT),withtheultimatepurposetoachieve neuroprotectionagainstcerebralischemia/hypoxia.

2.3. Surgicalproceduresandcollectionofsamples

For endogenous DMT measurements, rats were injected intraperitoneally(i.p.)withpargilin(50mg/bwkg,Sigma-Aldrich, USA),aneffectivemonoamine-oxidase(MAO)inhibitor,adayprior toandonthedayoftheexperiments.Forthesurgicalprocedures, animalswereanaesthetisedwith1.5–2%isoﬂuraneinN2O:O2(3:2) andwereallowedtobreathespontaneouslythroughaheadcone duringtheexperiment.Bodytemperaturewaskeptat37.2^◦Cby afeedback-controlledheatingpad(HarvardApparatus,Holliston, MA,USA).Atropine(0.1%,0.05mL;i.m.)wasadministeredaspre- medicationinordertoavoidtheproductionofairwaymucus.The leftfemoralarterywascannulatedfor blood sampling,andthe adjacentfemoralveinwascannulatedfordrugadministration.For thelaterinitiationofincompleteglobalforebrainischemia(n= 4),a midlineincisionwasmadeintheneck,andeach common carotidarterywascarefullyloopedaroundwithasurgicalthread.

Lidocaine(1%)wasusedforlocalanaesthesia.Controlratswere sham-operated(n=3).Allratswereﬁxedbytheirheadinaprone positioninastereotaxicapparatus.

After a baseline period (5–10 min), cerebral ischemia was inducedbytheocclusionofbothcommoncarotidarteries.Inhalf

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anhourafterischemiaonset,theinsultwasaggravatedbyhypoxia achievedbythecompletewithdrawal (5min,endogenousDMT measurement)orcontrolledwithdrawal(1min,exogenousDMT measurement)ofO₂fromtheanaestheticgasmixture.Thecom- plete withdrawal of O2 terminated the experiments,while the controlledwithdrawalofO₂wasfollowedbyreoxygenationandthe promptreleaseofthecarotidarteriestoallowcerebralreperfusion foranotherhour.

InratspreparedforthemeasurementofexogenousDMT,the administrationofDMT(1mg/bwkg/hinphysiologicalsaline)or its vehicle (physiological saline) started upon ischemia induc- tionthrough thefemoralvein, withtheaidof amicroinjection syringepump(CMA/100MicroInjectionPump,CarnegieMedicine, Stockholm,Sweden).Thei.v.infusionofDMTwascontinuousand maintaineduntiltheendoftheexperiment.

ForthedeterminationoftheendogenousDMTcontentofblood plasmaandbraintissue,5mLarterialbloodwascollectedimme- diately after the 5-min withdrawal of O2. The full blood was centrifugedpromptlyinanEppendorftube(5000g,5min,4^◦C, HeraeusFresco17Microcentrifuge,ThermoScientiﬁc,Waltham, MA,USA)coatedwithEDTA(ethylenediaminetetraaceticacid),and supernatantplasma(1mL)wasseparatedintoanotherEppendorf tube.Thebrainswereremovedandthen,aftersnap-frozeninliq- uidnitrogen,werestoredtogetherwiththeplasmaat−80^◦Cuntil furtherprocessing.

Forthemeasurementofbloodplasmaandbraintissueconcen- trationofexogenousDMT,bloodsampleswerecollectedthrough thefemoralarteryatbaseline(i.e.priortoischemiaonset),under ischemia,afterhypoxiaandunderreperfusion.Theanimalswere transcardiallyperfused withice-cold physiological salineunder deepchloralhydrate anaesthesia(5%,i.p.,500mg/bwkg)atthe endoftheexperimentalprotocoltoeliminateanybloodfromthe brain.Thebrainswereremovedandsnapfrozeninliquidnitrogen, thenstoredat−80^◦Cuntilfurtherprocessing.Bloodsampleswere processedasdescribedaboveandyielded0.2mLplasma.

2.4. Samplepreparationofratplasmasamples

Priortoextractionofplasma,5␮LIS(200ng/mLinmethanol) and5␮Lmethanolwasaddedto50␮Lofplasmasample.Aftervor- tex,50␮Lof5.3v/v%ammoniasolutionand300␮Lethylacetate wereaddedfollowedby30svortexandshakingfor10minatroom temperature(250rpm,orbital,Dual-ActionShakerKL2,Edmund Bühler,Bodelshausen,Germany).Thenthemixturewasrestedfor 5minonice.Thesamplewascentrifugedat4^◦Cfor12minat 21,000g(Universal320R,Hettich,Tuttlingen,Germany).250␮L ofthe upperphase wascollectedand thelowerphase wasre- extractedwith300␮Lofethylacetate.Aftercentrifugation,300␮L oftheupperphasewascombinedwiththeﬁrstportionofaliquot andwasdriedundernitrogenatroomtemperature.For2D-LC- HRMS/MSmeasurements,thedriedextractsweredissolvedin100

␮Lmethanol.

2.5. Samplepreparationofratbrainsamples

Rat brain tissue was homogenized based on the work by Kärkkäinen[10].Theweighedbrainsample(1gtissue,7mLice- cold0.1MHClO₄)wasaddedinto12mLtubes.Thesamplewas sonicatedonicewithBiologicsModel150VTultrasonichomog- enizer(BioLogics,Inc.,Manassas, VA,USA) for3 × 1minusing mediumpowersettingonthemicro-tipprobeandan80%pulse.

Aftercentrifugationat9000gfor12minat4^◦C(Universal320R, Hettich,Tuttlingen,Germany),5␮LIS(200ng/mLinmethanol), 5␮Lmethanolandﬁnally200␮Lof8%ammoniasolutionwere addedto400␮Lbrain homogenate.Aftervortex,500␮L ethyl acetatewasadded,followedby30svortexandshakingfor10min

atroomtemperature(250RPM,orbital,Dual-ActionShakerKL2, EdmundBühler,Bodelshausen,Germany).Thenthemixturewas restedfor5minonice.Thesamplewascentrifugedat4 ^◦Cfor 12minat21,000g(Universal320R,Hettich,Tuttlingen,Germany).

350␮Loftheupperphasewascollectedandthelowerphasewas re-extractedwith500␮Lofethylacetate.Aftercentrifugation,500

␮Loftheupperphasewascombinedwiththeﬁrstportionofthe aliquotfollowedbydryingundernitrogenatroomtemperature.

Forthe2D-LC-HRMS/MSmeasurements,thedriedextractswere dissolvedin100␮Lmethanol.

2.6. Preparationofsamplesforcalibrationandvalidation

1.00mg/mL stocksolutionsofDMTand AMTwereprepared by dissolving approximately1 mg aliquots of each component inmethanol.Thestockswereusedforthepreparation ofwork- ingcalibrationstandards.EightDMTworkingcalibrationstandard solutionswerepreparedinmethanol.Forexternalcalibration,the sample preparation wassimilar tothat of thetreated samples, exceptthatthesamplewasspikedwith5␮Lofthegivenconcen- trationofDMTinsteadof5␮Lmethanol.Thus,theﬁnalvolumes werethesameinthecalibrationsamplesandthetreatedsamples.

Externalcalibrationcurvesusinginternalstandardwereusedwith thefollowingcalibrationpointsofDMT(0,0.2,2,10,20,40,100 and200ng/mLplasma)inplasmaand(0,0.175,1.75,8.75,17.5,35, 87.5,175and350ng/g)inbrain.Eachcalibrationpointwasinjected inreplicateandrepeatabilitywasevaluatedfortheretentiontimes ofDMT.

Thelimitofdetection(LOD)andlimitofquantiﬁcation(LOQ) werecalculated,respectively,onthebasisonthestandarddevia- tion(SD)andtheslope(S)ofthecalibrationcurve.

LOD= 3.3×SD S

LOQ= 10×SD S

Accuracyandprecisionwasdeterminedbyanalysingsamples at“Low”(10ng/mLinplasmaand17.5ng/ginbrain),“Mid”(40 ng/mLinplasmaand35ng/ginbrain)and“High”(200ng/mLin plasmaand350ng/ginbrain)concentrationsinthreeanalytical runs.

Carryoverwasassessedbyinjectingablanksampleafterinject- ingahighconcentrationofanalyte(plasma:200ng/mL,brain:350 ng/g).

ToevaluatethestabilityofDMTinplasmaandbrainsamplesin theautosampler(16^◦C),calibrationsolutions(20ng/mLinplasma and35ng/ginbrain)werere-injected24haftertheﬁrstinjec- tionandcomparedtotheoriginalconcentrations.Theresultsofthe secondrunswereexpressedasthepercentageoftheirrespective valuesintheﬁrstrun.

2.7. 2D-LC-HRMS/MSconditions

The 2D-LC-HRMS/MS analysis was performed with Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap (Thermo FisherScientific,Waltham,MA,USA)massspectrometercoupled to a Waters Acquity I-Class UPLC^TM (Waters, Manchester, UK) apparatus.TheUHPLCsystemequippedwithtwobinarysolvent managers,anauto-samplerandacolumnmanagerwithtwosix- port,two-positionautomaticswitchvalves.Theeluentofthetrap columnwasdilutedusingaKnauerHPLC-pump64(Knauer,Berlin, Germany)andahigh-pressurestainlesssteeltee(IDEX,OakHarbor, WA,USA).Thefirst-andsecond-dimensionalanalyticalcolumns werethermostattedinthecolumnmanageroftheUHPLCsystem,

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whilethetrapcolumnwasinsertedintoanl-7350LaChromcolumn oven(Merck,Darmstadt,Germany).

In the ﬁnal 2D-LC-HRMS/MS analysis, the ﬁrst-dimensional chromatographicseparation wascarried outonaKinetexHILIC column(150×2.1mm,2.6␮m,100Å,Phenomenex)protected byaHILICguardcolumn(4×3mm,2.6␮m,100Å,Phenomenex), whilethesecond-dimensionalseparationwasperformedonaLuna Omega Polar C18 column (50 × 2.1 mm, 1.6 ␮m, 100 Å, Phe- nomenex).For theenrichmentof theanalytes,aLunaC18trap column(20×2mm,5␮m,100Å,Phenomenex)wasappliedand connectedthroughtwosix-portvalves(Fig.1).

Duringmethoddevelopment,thefollowing chromatographic columnsweretestedin theﬁrstdimension:LunaHILIC(150 × 3 mm,3 ␮m, 200Å, Phenomenex), LunaOmega Sugar (100 × 2.1 mm,3 ␮m, 100 Å,Phenomenex), LunaNH₂ (150 × 2 mm, 3␮m, 100Å,Phenomenex)andKinetexC18(50× 2.1mm,2.6

␮m,100Å,Phenomenex).Columnstriedintheseconddimension wereLunaOmega PSC18 (50 × 2.1 mm,1.6 ␮m, 100Å, Phe- nomenex),AcquityUPLCBEHC18(50×2.1mm,1.7␮m,130Å, Waters)andCORTECSUPLCC18+(50×2.1 mm,1.6␮m,90 Å, Waters).Bythe scheduledswitching ofthesix-portvalves,the dilutemobilephasefromtheHILICcolumninﬁrstdimensionwas trappedonLunaC18enrichmentcolumn(Fig.1).Forthepreven- tionoftheelutionoftargetedcompoundsfromtheenrichment columnsduringthetrappingprocess,thediluterpumpdelivered aqueousammoniasolution(pH10.2)at3mL/minﬂowrateinto thestaticmixingtee,whichwasconnectedtoaHILICcolumnand asix-portvalve.Thetemperatureoftrapcolumnswasmaintained at50^◦C(Fig.1).

Intheﬁrstdimension,a10␮Lsamplewasinjectedwiththe Partial LoopWith Needle Overﬁll injection modeon a Kinetex HILICcolumnusingtheprogrammedgradientofeluentA(50mM ammoniumformatesolution)andeluentB(0.1%formicacidin acetonitrile).Thecolumntemperaturewassetat50^◦Cduringthe analysis.Inthesecond dimension,eluent AandB,respectively, were0.1%formicacidsolutionand0.1%formicacidinacetonitrile.ThemobilephasewashedthesubstancestrappedonaLuna C18enrichmentcolumnontoaLunaOmegaPolarC18column.The second-dimensionalcolumnwasmaintainedat50^◦C.Thegradient programsofbothdimensionalseparationswiththetwosix-port valvepositionsaredetailedinTable1.

The mass spectrometer was operated in positive-ion mode usinga heatedelectrosprayionisation(ESI)sourcewiththefol- lowingconditions:capillarytemperature250^◦C,S-LensRFlevel 50,sprayvoltage3.5kV,sheathgasflow45,sweepgasflow2and auxiliarygasflow10 inarbitraryunit.Inparallelreactionmon- itoring(PRM)withresolutionof17,500(FWHM),theautomatic gaincontrol (AGC)settingwasdefined as1 × 10⁶ charges and themaximuminjectiontimewassetto50msThewidthofthe isolation windowof precursor ion was2 Da. Collisionenergies (CE)wereoptimisedforrespectivetransitionsofDMTandAMT (Fig.2).TheadditionalmainHRMS/MSparametersaresummarized inTable2.

During2D-LC-HRMS/MSmeasurements,theeluatewaspassed intotheESIsourceonlyinthetimerangeof4.5–5.5mininorder todecreasethecontaminationoftheMS.Fortheremainingtime, theESIsourcewasrinsedwithacetonitrile/watersolution(90/10, v/v)ataﬂowrateof0.1mL/minbyanauxiliary pump(Knauer HPLC-pump64,Knauer,Berlin,Germany).

TheLCsystemwascontrolledbyMassLynx4.1SCN901(Waters, Milford,MA,USA).ThecontroloftheMSsystemandMSdataacqui- sitionwasconductedbyXcalibur^TM4.1software(ThermoFisher Scientiﬁc,Waltham, MA).GraphPad Prism5statistical software (GraphPadSoftware,SanDiego,CA,USA)wasusedfordrawingcol- umndiagrams,boxplotsandpointgraphs,andthecalculationof pairedt-test.

3. Resultsanddiscussion

3.1. Developmentofheart-cutting2D-LC-HRMS/MS

During analytical method development several parameters weretestedinbothdimensionsandtrappingproceduresuchas mobilephasecomposition,columntemperature,gradientsteep- ness, flow rate, type and length of column. For ESI-HRMS/MS detectionofDMTandAMT,theappropriatetransitions(quantifiers, qualifiersions)wereselectedandtherelatedcollisionenergiesand ESIparameterswereoptimised(Fig.2).

ThemainaimofoptimisationofHILICseparationofDMTand AMT(internalstandard)wastoachieveproperretainand peak shapeofDMTandAMT,whichisfeasibleinvolvingequilibration timewithinmaximum10minasaplannedcompleteruntimeof online2Dmeasurements.FortheoptimisationofHILIC,narrow- borecolumns(3and2.1mm)withlengthof100mmand150mm, packedwithtotallyporousorcore-shellparticleswerecompared withdifferentsolidphases.Thepresenceofwaterataminimum quantityof3%intheinitialmobilephasecompositionisessential inHILIC.Byvaryingitsconcentration,theretentionmechanismand separationcanbeinﬂuencedinbothisocraticandgradientelutions [19].

TheMS-compatibleammoniumformateasmobilephasemod- ifierwasselectedat50mMconcentrationapplyingacetonitrileas theorganiccontentofthemobilephase.Theflowrateisalsoacrit- icalparameterinthefirstdimension,sinceitmustbekeptaslow aspossible,thusassistingthetrappingoftheinvestigatedanalytes.

Unfortunately,theundesiredeffectoflowﬂowrateispeakbroad- ening.Inourcase,aﬂowrateof0.3mL/minprovidedanacceptable compromise.Forappropriatecolumnselection,fourHILICcolumns weretested(LunaHILIC,KinetexHILIC,LunaOmegaSugar,Luna NH₂)usinga50mMammoniumformateaqueoussolution/0.1% formicacidinacetonitrile(90/10,v/v)mobilephasecomposition for6min.AcomparisonoftheseparationperformanceofAMTand DMTonthefourHILICcolumnsrevealedthatapplicationofLuna Omegawasdisadvantageousforbothretentionandpeakshapes ofDMT(1.35min)andAMT(1.40min).Similarly,whenLunaNH2

wasused,AMTgaveanextremepeakbroadening,whereasDMT (1.23min)hadarelativelylowretention.InthecaseofLunaHILIC, signiﬁcantlyhigherretentiontimeswerefoundforbothDMT(3.89 min)andAMT(4.84min)withapeakwidthofapproximately1 min.Interestingly,theelutionorderchangedusingKinetexHILIC column,andtheretentiontimesofbothanalyteswerehigherthan 6 min.Therefore, theeluent strength wasincreased to50 mM ammoniumformateaqueoussolution/0.1%formicacidinacetonitrile(85/15,v/v)resultingin3.09minand2.72minretentiontime forDMTand AMTand a peakwidthof approximately0.5min.

Consequently,thiscompositionwasselectedintheﬁnalmethod.

Interestingly,theelutionorderchangedusingtheKintexHILICcol- umnincomparisonwiththoseoftheotherthreecolumns.

Inthecaseofoptimisationofseconddimension,theprimary aimwastoobtainsharp,symmetricchromatographicpeaks(high sensitivity)withcloseretentionsforbothcompoundsusinggra- dientelutionandkeeping 10mintotalruntimeof2Danalysis.

Initially,ﬁveC18columnswerecompared,usinggradientelution (0–1min10%B,1–4min40%B,whereeluentsAandB,were0.1

%formicacidaqueoussolutionand0.1%formicacidinMeOH), respectively.UsingCORTECSC18,OmegaPSC18,andKinetexC18, relativelyhighpeakwidths(∼0.5and0.3minforDMTandAMT) wereobserved.TheapplicationofBEHC18andOmegaC18pro- videdhigherretentiontimesandlowerpeakwidthscomparedwith thoseoverotherC18columns.Finally,anOmegaC18microbore columnwithaparticlesizeof1.6␮mwasselectedforfurtheropti- misationprocedures.Thenatureoforganicsolventinﬂuencedboth peakwidthandretention.ForDMTandAMT,itcouldbeconcluded

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Fig.1.Flowschemeoftheestablished2D-LC/MSsysteminbothvalvepositions:Position1(A)andPosition2(B).

Fig.2. ThestructureofDMT(A)andAMT(B)withassumedfragmentationcleavages,theexactmassofquantiﬁerandqualiﬁerionsandtheiroptimisedcollisionenergy.

thatACNinsteadofMeOHgavelowerretention,betterpeakshape andlowerpeakwidth.Forobtainingproperretentionwithin10- minruntimeincludingwashingandre-equilibrationofthecolumn

connectedtotrappingcolumnintheseconddimension,thetem- peratureofthecolumn,theﬂowrateandthegradientsteepness wereadjustedandsynchronizedwiththeHILICmethod.

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Table1

Thegradientprogramsofﬁrstandseconddimensionwiththetwosix-portvalvepositionsandanalysismode.

Gradientprogramof1^stdimension Gradientprogramof2^nddimension Timetableofvalves Mode Time(min) B% Flowrate(mL/min) Time(min) B% Flowrate(mL/min) Time(min) Valve1 Valve2

0.00 85 0.3 0.00 2 0.5 0.00 Position2 Position2

1.80 Position1 Position1

trapping

3.00 2 0.5

3.10 2 0.3

3.50 2 0.3 3.50 Position2 Position2

4.00 85 0.3

analysis

4.50 50 1.0

5.50 100 0.3

6.00 100 0.3

6.10 100 0.5

6.50 50 1.0

7.00 85 1.2

8.00 Position1 Position1 8.01 Position2 Position2

9.00 100 0.5

9.50 2 0.5 9.50 Position1 Position1

9.51 Position2 Position2

9.90 85 1.2

10.00 85 0.3 10.00 2 0.5

Table2

MainHRMS/MSparametersforDMTandAMT(internalstandard)andretentiontimes.

Analyte Chemical formula

[M+H]⁺ Production1 (quantiﬁerion)m/z

Collisionenergy (eV)

Production2(qualiﬁer ion)m/z

Collisionenergy (eV)

Retentiontime (min)

DMT C12H16N2 189.1386 58.0651 10 144.0808 21 4.99

AMT C11H14N2 175.1230 158.0964 10 143.0730 30 4.99

Theonlinetrappingof DMTand AMTfromtheeluateofthe ﬁrst-dimensionalHILICcolumnina2D-LCsystemwasperformed byusingC18trapcolumn.Thishighlyorthogonalseparation(HILIC

×RP-LC)requiredtodecreasethesolventstrengthoftheeffluentof thefirst-dimensionalcolumninordertoretainbothanalytesonthe enrichmentcolumn.Fig.1.demonstratestheexperimentalconfig- urationofour2D-LC-HRMS/MSsystem.The2Dinterfacebetween HILICandRPcolumnsconsistsofC18trapcolumnandtwoauto- maticallyoperatedtwo-positionsix-portvalves.Thedilutionofthe first-dimensionalHILICeluatewasperformedbyapplyingadiluter pumpconnectedtothecolumnthroughastaticmixingtee(Fig.1).

Theselectionofanappropriatedilutersolventandflowratewas crucialtoenrichDMTandAMTonthetrappingcolumnwithhigh efficiency.Itcanbeexplainedbythehighelutingpowerofeluate ofHILICseparationinRPmodeand,consequently,itneededtobe dilutedwithaweakRPsolvent.Initially,0.1%formicacidaque- oussolutionwasusedwithaflowrateof1mL/min.However,it resultedinpoorretentionforbothanalytesonthetrappingcolumn (Fig.1).Therefore,theeffectoftheflowrateofthedilutingeluenton retentionwasinvestigatedintherangeof1–3mL/min.Fig.3shows that,byincreasingtheflowrateupto3mL/min,trappingefficien- cieswereonlyslightlyimproved.Thesenegligiblechangescanbe explainedbythepKavaluesofDMT(8.68)[20]andAMT(9.96)[21].

AcomparisonrevealedthatatlowretentionintheRPmodeusing of0.1%formicacidaqueousdilutersolutionwithapHof2.7,the protonatedformsofDMTandAMTwerethedominantspecies.It seemedobviousthattheretentionofAMTandDMTonthetrapping columncanbeenhancedbysuppressingthepolarcationicforms byincreasingthepHofthedilutersolvent.TheincreaseofpHupto 10.2inaqueousammoniasolutionwasanacceptablecompromise consideringthesufﬁcienttrappingofthetwoanalytesandthepH stabilityofboththetrappingandtheOmegaC18columns.Fig.3 demonstratesthedramaticimprovementoftrappingefﬁciencyof DMTandAMTbytheincreaseofpHofthedilutersolvent.ForDMT, higherenhancementwasfound,whichcouldbeexplainedbyits lowerpKavalueby∼1.3unitscomparedwiththatofAMT.

Fig.3.TheeffectoftheﬂowrateofdilutingeluenttotrappingefﬁciencyofDMT andAMTduringtrappingoftheheart-cut1Deluateintherangeof1–3mL/min (0.1%FA),and3mL/min(ammoniasolutionatpH10.2).Thepercentageincreases comparedtoconsecutiveexperimentsareindicatedbycurvedarrows.

Fig. 1 (A) displays the schematic diagram of our 2D-LC- HRMS/MSsystemin ¨Position1status,wherethe ¨C18Trap¨column wasusedtotrapDMTandAMTfromthediluteeffluentoftheHILIC column.Duringthetrappingperiod,thegradientprogramofHILIC methodwasinisocraticsection(Table1).Thesystemwaskept intrappingconfigurationfrom1.8minto3.49min.At3.50min theswitchingvalveswereturnedinto ¨Position2andthus ¨C18 trap¨columnwasconnectedtotheRPanalyticalcolumnasshown inFig.1(B).Theparticulardesignoftheeluentgradientprogram wasappliedtowashouttrappedanalytesfrom ¨C18trap¨column ontoC18analyticalcolumn,wherethechromatographicseparation occurredfollowedbyHRMS/MSdetectionofDMTandAMT.Table1 summarizesthefinalsetupofeventsandgradienttimeprograms inbothdimensions.Tokeeptheshort10min2Druntime,theHILIC columnwaswashedandequilibratedfrom3.5to10min,whilethe

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Fig.4. ExtractedionchromatogramsofquantiﬁeriontransitionofDMTandAMTdetectedbypositivemodewith1D-HILICmethod(A)and2D-LC-HRMS/MSmethod(B)in standardsolution.

trapandC18columnswereintherangesof8–10minandupto3.5 mininthenextrunusingeluentandtheflowrategradientpro- graminbothdimensions.Theobtained10mintotalruntimewas fasterthanvariouspublishedonedimensionalLC–MS/MSmethods forDMTanalysisinbrainandplasma[15–18].Theelutionprofiles ofDMTandAMTinthefinal1Dand2Dchromatogramsofstandard solutionsareshowninFig.4(A,B).

3.2. Determinationofextractionrecovery,matrixeffect,process efﬁciencyandmainvalidationparameters

Hence,thechallengeofthisworkwastofindarelativelysim- plesamplepreparationprocedure,whichissuitableforenrichment ofDMTfromratplasmaandbrainsamples.Intheliterature,several sample preparation methods related to DMT are reported [10,15–18,22,23].Unfortunately,inmanycasesthemainparam- eters,suchasextractionrecovery(RE),matrixeffect(ME),process efficiency(PE),characteristicsoftheefficiencyofagivenmethod, werenotpresented.Labour-intensiveandexpensivesolid-phase extraction procedures are widely employed nowadays for the enrichmentofDMTfrommammaliantissues[10,15,16].Theappli- cationof2D-LChasanabilitytodecreasethenegativematrixeffect underanalysistherebyprovidinganopportunitytosimplifythe samplepreparationprocedure.Therefore,theliquid–liquidextrac- tionprocedureseemedtobeanobviouschoiceforthedevelopment ofanewmethod.Withoutdetailingtheprocessoptimisationofthe samplepreparationprocedure,theuseofethylacetateasorganic solventandabasicpHinsamplesolutionhelpedtoenrichDMT properlyinthecaseofplasmaandbrainsamples(Fig.5(A,B)).

TherelevantRE,MEandPEofthenewmethodweredeterminedin accordancewiththeprocedureofMatuszewskietal.andCappiello etal.[24,25]

Fig.5(A,B)illustratesthemainindicators(RE,MEandPE)of thenewanalyticalmethodforDMTandAMTinratplasma(20 ng/mL)andbrain(17.5ng/mL).ForDMT,theobtained90%ofRE ofournewsample preparationprocedureinplasmawasbetter withapproximately20–30%thandatainearlystudies[17,18].The obtainedhighmeanvaluesofREwithreallygoodrepeatability(∼

Table3

Mainvalidationparametersof2D-LC-HRMS/MSmethod.

Plasma Brain

Repeatabilityofretentiontimerange(min) 4.99±0.01 4.99±0.01

LOD 0.108ng/mL 0.212ng/g

LOQ 0.328ng/mL 0.642ng/g

AccuracyLow(%) 111.7 111.6

AccuracyMid(%) 89.6 88.5

AccuracyHigh(%) 102.6 100.9

PrecisionLow(%CV) 2.8 2.7

PrecisionMid(%CV) 1.4 5.1

PrecisionHigh(%CV) 1.4 1.3

Carryover(%) 0.056 0.078

Stability(%) −0.452 −0.500

Limitofdetection(LOD),limitofquantiﬁcation(LOQ)andpercentcoefﬁcientof variation(%CV).

10%SEM)forDMTandAMTstandardrevealthatournewextrac- tionmethodsarewell-suitedforbothplasmaandbrainsamples.

ThepresenceofstrongionisationenhancementduringHRMS/MS detectionresultedinahighpositiveMEvalueofDMTfromplasma (161%)andbrain(174%).TheobtainedhighMEinplasmawas much favourablethan reportedbyMeyers (46%) in 2014[17].

However,byinvestigatingMEofAMT,moderateionsuppression occurredresultinginavalueof87%inbrain(Fig.5(B)).Overall,the obtainedfavourableMEvaluesforbothcompoundsfromacomplex biologicalmatrixclearlyindicatedthebeneﬁtofapplicationofthis 2D-LC-HRMS/MSmethod.InthecaseofDMT,the145%(plasma) and153%(brain)ofPE,asresultantsofvaluesofREandPE,lend evidencetotheadequacyofthenewmethod(Fig.5(A,B)).

Forbrainandplasmasamples,themainvalidationparameters ofDMTaresummarizedinTable3.Afterfulloptimizationofthe2D method,goodrepeatabilityoftheretentiontimeofDMTwithinthe rangeof4.98–5.00minwasachieved.Duringmethoddevelopment, themainissuewastokeeptherequiredvolumeofplasmaaslow aspossibleinordertodecreasethenegativeeffectofbloodcollec- tionduringsurgicalprocedures[26].ForDMT,our2D-LC-HRMS/MS methodprovided0.108ng/mLofLODusing50␮Lofplasma,which issigniﬁcantlybetterthanthosereportedinearlierstudies,suchas

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Fig.5.Extractionrecovery,matrixeffectandprocessefﬁcienciesforDMT(plasma:

20ng/mL,brain:17.5ng/mL)andAMT(plasma:20ng/mL,brain:17.5ng/mL)stan- dardsfromratplasma(A)andratbrainhomogenate(B).Theextractionrecoveryand matrixeffectpercentagewerecalculatedasfollows:Extractionrecovery(%)=C/B

×100,Matrixeffect(%)=B/A×100andProcessefﬁciency(%)=C/A×100,whereA representstheaveragepeakareaofthestandardsolution,Brepresentstheaverage peakareaoftheplasmaorbrainextractspikedatthesameconcentrationofthe standardandCrepresentstheaveragepeakareaofaplasmaorbrainpre-spikedat thesameconcentrationofthestandard.

100ng/mLusing500␮Lplasma[17]and0.45ng/mLfrom200␮L plasma[18].Interestingly,Oliveiraetal.reported0.1ng/mLofLOD forDMTinplasma[16].However,thesolidphaseextractionpro- cedureoftheiranalyticalmethodrequired1000␮Lofplasma.In brainsampletheachievedLODofDMTinourcasewasalsobetter thanthatreportedbyBarkeretal.(0.5ng/g)[15].

Thecalibrationcurves(Figs.S13–S24)forDMTwerelinear(R2

>99%)intherangeof0.2–200ng/mLplasmaand1.75–350ng/g brain.Accuracyofournewmethodinthreedifferentconcentration levelsweredeterminedwithinanacceptableintervalof12%in plasmaandbrainsamples.Theprecisionswerefoundtobe≤2.8% coefﬁcientofvariation(CV)incaseofplasmaand≤5.1%CV(brain).

TheobtainedcarryoverofDMTwasnegligible(<0.08%)inplasma andbrain.

3.3. QuantiﬁcationofDMTinratplasmaandbrain

Intheﬁrstdimensionofourorthogonal2Dmethod,HILICmode was appliedfor theseparation of DMT and AMT from plasma andbrainmatrix-derivedendogenouscomponents.Inthemethod developmentprocess,animportantconsiderationwastocutthe peaksofinterest from1Dto2Din anarrowtime window(1.8 minfor 3.5min), thus tokeeptheamountof undesirable exo- andendogenousmatrixcompoundsinalowlevelinthesecond dimension.Itisworthtobenotedthatthetrappingprocessatbasic pHalsodecreasedtheamountofcompoundswithacidiccharac- terwithachromatographicbehavioursimilartothoseofDMTand AMTinHILICcondition.Forthesereasons,arelativelyfastgradient methodwasappliedintheseconddimension.TheobtainedMEval- uesconﬁrmedthevalidityofthesteepRPgradientprogramapplied.

AnotheradvantagewasthatAMTasinternalstandardallottedwith DMTastargetedcompoundatsameretentiontime,butnotindead time,whichisthechromatographicexpectationofthestandard calibrationmethodtowardsaninternalstandard(Figs.S1–S4).

ThedevelopedmethodwasappliedtothequantificationofDMT inratplasmaandbraintissue.Themainaimsoftheapplication ofthisnew2D-LC-HRMS/MSmethodweretofollowtheplasma DMTlevelbeforeischemiaonset, underischemia,afterhypoxia andunderreperfusion,anddeterminationofDMTconcentration inrelatedbraintissueafterreperfusion.Therefore,theplasmaand brainsamplesofpreliminaryexperimentswereusedforoptimi- sationofthesamplepreparationprocedureanddeterminationof theconcentrationrangeofexternalcalibrationusinginternalstan- dards.Awidecalibrationconcentrationrangewasattemptedfor quantificationofendogenousandexogenousDMTlevelsinasingle run.Toourknowledge,thereexistsonlyasinglereportfrom2005 inwhichendogenousDMTwasquantifiedinbothratbrainand plasmausingisoctraticLC–MS/MSmethod[10].Unfortunately,we couldnotconfirmthepresenceofendogenousDMTinratbrainand plasma.Itisimportanttonote,however,thatour2D-LC-HRMS/MS methodprovidedmorereliabledatadueto2D-LCseparationand accuratehighresolutionmassspectrometrydetectionwithrelated strictLCandHRMSMSconfirmatorycriteria:specificretentiontime (4.99min)withtwocharacteristicprecursor–productiontransi- tionsusing50ppmmassaccuracy(quantifierionat58.0651m/z andqualifierionat144.0808m/z)andtheratioofthepeakarea ofthequantifierionandqualifierion(2.1±0.21)(Figs.S5–S12).

Despitethefactthatthequantiﬁerionwasdetected,butonethe above-mentionedotherparameterswasnotcompleted,thecon- centrationofDMTwasfoundtobelowerthanthelimitofdetection.

Intheﬁnalmethod,thecalibrationcurvewasoptimisedtomea- sureexogenousDMTlevel,andDMTshowedagoodlinearityrange of0.2–200ng/mLplasmaand1.75–350ng/gbrainwithR2values higherthan0.99(Figs.S13,S14).

ThelevelofDMTinplasmasamplesoftreatedratsisillustrated inFig.6(A).Allplasmasamplescollectedbeforeadministrationof DMTshowedlessthanthedetectableamountsofDMT.Thehigh- estconcentrationofDMTintherangeof49.3–114.3ng/mLwas observedinplasmaforeachanimalbeforehypoxia.Foranimals1, 3and4,theconcentrationsofDMTdecreasedsigniﬁcantlyafter hypoxiainplasma.Afterhypoxiatotheendofthetreatment,the concentrationofDMTdecreasedtoameanvalueof3.8ng/mL,in linewiththeterminationofDMTapplication.

Another question that remains to be answered is whether DMTaccumulatestoadetectableconcentrationinthebrain.Thus, endogenousandexogenousDMTwereanalysedinbraintissuein boththecontrolandtreatedanimals.Similartoplasmasamples, theendogenousDMTwasnotdetectableinthebrainofcontrolani- mals.However,aftertreatment,theconcentrationofDMTchanged intherangeof2–6.1ng/gbrain(Fig.6(B)).

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Fig.6. TheobtainedconcentrationofDMTinplasma(A)andbrain(B).Three repeatedmeasurementswereusedforstatisticalanalysis(*p≤0.05;**p≤0.01;***

p≤0.001).

Theconcentrationsof exogenousDMTmeasuredareconsid- eredtobeunderestimatedoftheactualconcentrations,because DMTmusthavebeenrapidlydegradedduringcollectingbiological samples[27],inparticular,whenharvestingbraintissue.

4. Conclusion

Thispaperdescribesthedevelopmentandapplicationofafast heart-cutting2D-LC-HRMS/MSmethodand relatedliquid-liquid extractionprocedurefortheanalysisofDMT.Byconnectingorthog- onalHILIC and RPchromatography through theuseof RP trap column and high resolution MS/MS detection, a sensitive and selectiveanalyticalmethodwassuccessfullyutilisedwithintotal run time of 10 min for determination of the concentration of DMTinratplasmaandbrainofexperimentalmodelofcerebral ischemia/reperfusion using DMT administration The developed andoptimisedplasmaandbrainmatrix-relatedsampleprepara- tionprotocolsprovided high extractionrecoveries(∼90 %) and highpositive matrix effect (plasma: +61 %, brain: ∼ +74 %) of DMT.During analysis,DMTwasidentifiedandconfirmedbyits specificretentiontime inthesecond dimension,twocharacter-

isticprecursor–productiontransitionsandtheratioofquantiﬁer toqualiﬁer masstransitions. External calibrationusinginternal standard wasappliedtoobtainaccurateconcentrationsof DMT inplasmaandbrainsamples.Thisheart-cutting2D-LC-HRMS/MS methodwithnewsamplepreparationproceduregaveanopportu- nitytoimprovetheLODwiththedecreaseoftherequiredamount ofbiologicalsamples.

ConcerningexogenousDMT,tothebestofourknowledge,thisis thefirstattempttoconfirm–withananalyticaltool–thepresence ofDMTadministeredwiththerapeuticalpurposeinmammalian tissues.Insummary,ouranalyticalapproachissuitabletodetect andconfirmthepresenceofDMTadministeredtoexperimental animalswiththerapeuticpurpose.Furtherrefinementofbiologi- calsampleharvestingwillprovidemoreaccuratedeterminationof actualDMTconcentrations.

DeclarationofCompetingInterest Authorsdeclarenoconﬂictofinterest.

Acknowledgement

This research was supported by the EU-funded Hungarian grantEFOP-3.6.1-16-2016-00008 and <GN1>G</GN1>INOP-2.3.2- 15-2016-00060,andtheMinistryofHumanCapacitiesofHungary grantNo.ÚNKP-19-3-SZTE-266.

WewishtothankÉvaSija(InstituteofForensicMedicine,Uni- versityofSzeged),whokindlyprovidedtheAMTstandard.

AppendixA. Supplementarydata

Supplementarymaterial related tothis articlecan befound, in theonline version, at doi:https://doi.org/10.1016/j.jpba.2020.

113615.

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