Parallel artificial liquid membrane extraction of new psychoactive substances in plasma and whole blood

ContentslistsavailableatScienceDirect

Journal of Chromatography B

j o ur na l h o me pa g e :w w w . e l s e v i e r . c o m / l o c a t e / c h r o m b

Parallel artificial liquid membrane extraction of new psychoactive substances in plasma and whole blood

Linda Vårdal

, Hilde-Merete Askildsen

, Astrid Gjelstad

, Elisabeth Leere Øiestad

, Hilde Marie Erøy Edvardsen

, Stig Pedersen-Bjergaard

aSchoolofPharmacy,UniversityofOslo,P.O.Box1068,Blindern,0316Oslo,Norway

bNorwegianInstituteofPublicHealth(NIPH),DepartmentofForensicSciences,OsloUniversityHospital,Oslo,Norway

cFacultyofHealthandMedicalSciences,SchoolofPharmaceuticalSciences,UniversityofCopenhagen,Universitetsparken2,2100Copenhagen,Denmark

a r t i c l e i n f o

Articlehistory:

Received15December2016

Receivedinrevisedform8February2017 Accepted10February2017

Availableonline14February2017

Keywords:

Liquid-phasemicroextraction Parallelartificialliquidmembrane extraction

Newpsychoactivesubstances Plasmasamples

Wholebloodsamples

a b s t r a c t

Parallelartificialliquidmembraneextraction(PALME)wascombinedwithultra-highperformanceliquid chromatography-massspectrometry(UHPLC–MS)andthepotentialforscreeningofnewpsychoactive substances(NPS)wasinvestigatedforthefirsttime.PALMEwasperformedin96-wellformatcomprising adonorplate,asupportedliquidmembrane(SLM),andanacceptorplate.UnchargedNPSwereextracted fromplasmaorwholeblood,acrossanorganicSLM,andintoanaqueousacceptorsolution,facilitatedby apHgradient.

MDAI (5,6-methylenedioxy-2-aminoindane), methylone, PFA (para-fluoroamphetamine), mCPP (meta-chlorophenylpiperazine), pentedrone, methoxetamine, MDPV (methylenedioxypyrovalerone), ethylphenidate,2C-E (2,5-dimethoxy-4-ethylphenethylamine),bromo-dragonfly,andAH-7921 (3,4- dichloro-N-{[1-(dimethylamino)cyclohexyl]methyl}benzamide)wereselectedasrepresentativeNPS.

OptimizationofoperationalparameterswasnecessaryastheNPSwerenoveltoPALME,andbecause PALMEwasperformedfromwholebloodfortheveryfirsttime.InthePALMEmethoddevelopedfor plasma,NPSwereextractedfroma250␮Lalkalizeddonorsolutionconsistingof125␮Lplasmasample, 115␮L40mMNaOH,and10␮Linternalstandard.InthePALMEmethodfromwholeblood,the250␮L alkalizeddonorsolutionconsistedof100␮Lwholeblood,50␮Ldeionizedwater,75␮L80mMNaOH, and25␮Linternalstandard.Inbothmethods,extractionwasaccomplishedacrossanSLMof5␮Ldodecyl acetatewith1%trioctylamine(w/w),andfurtherintoanacidicacceptorsolutionof50␮L20mMformic acid.Theextractionwaspromotedbyagitationat900rpmandwascarriedoutfor120min.Methodval- idationwasperformedandthefollowingparameterswereconsidered:linearity,limitsofquantification (LOQ),intra-andinter-dayprecision,accuracy,extractionrecoveries,carry-over,andmatrixeffects.The validationresultswereinaccordancewithFDAguidelines.

©2017ElsevierB.V.Allrightsreserved.

1. Introduction

Newpsychoactivesubstances(NPS)areoccasionallyreferredto as“legalhighs”.Thistermhasreceivedcriticismasitmaybeincor- rectlyinterpretedbecauseoftheword“legal”[1].Still,thetermis basedonthefactthatNPSresembleothercontrolledsubstancesor

“illegalhighs”inmolecularstructureandpharmacologicaleffects, butmightnotbesubjectedtoregulatorycontrolthemselves[2].

TheprevalenceandtheusageofNPShasincreaseddramaticallythe last10–15years,andithasbecomeaseveresocialprobleminter-

∗Correspondingauthorat:SchoolofPharmacy,UniversityofOslo,P.O.Box1068, Blindern,0316Oslo,Norway.

E-mailaddress:[email protected] (S.Pedersen-Bjergaard).

nationallyasNPSrepresentamajorhealthrisk[3].Theincrease ismainlyduetoon-linesalesontheinternet,wheretheproducts arefrequentlypromotedasharmlesseverydayproducts,likebath salts[4].ThisisunfortunateasNPScanbemoretoxicandpotent thanthecontrolledsubstancestheyresemble.Inaddition,thephar- macologicaleffectmayhaveaconsiderablysloweronset.Thelatter mayconfusetheuserandleadtoahigherintakeinordertoachieve thedesiredeffectmorequickly,resultingintoxicityorworstcase scenario:fatality[5].Thedevelopmentandcreativitybehindthe productionandspreadingofNPSisgroundbreakingandhasputthe internationalsystemfordrugcontrolinapinch[6].Allinitiatives directedtowardscombattingtheNPSphenomenonisthereforeof greatimportanceandtheneedforsolidforensicanalyticalmeth- odstoachieveaccurateidentificationofnewsubstancesisobvious.

Intheliterature,liquidchromatography(LC-),gaschromatography

http://dx.doi.org/10.1016/j.jchromb.2017.02.010 1570-0232/©2017ElsevierB.V.Allrightsreserved.

techniquesinteresting andimportant,withspecialemphasison providinghighthroughput,extensiveclean-uppriortoLC–MS/MS, andasimplework-flow[12].

Parallel artificial liquid membrane extraction (PALME) was introducedin2013asanewmicroextractiontechniqueinthe96- wellformat[13].The PALME principle issimilar tothe closely relatedtechniquehollow-fiberliquid-phasemicroextraction(HF- LPME),whereapHgradientisusedtofacilitatemasstransferof unchargedanalytesacrossanorganicsupportedliquidmembrane (SLM).Thecommerciallyavailableequipmentthatwasusedfor PALMEinthispaperisdepictedinFig.1andiscomprisedbyadonor plate,anacceptorplate,andatoplid.Thedonorplatehousesan aqueousdonorsolution(sample)andtheacceptorplatehousesan aqueousacceptorsolution(PALMEextract).Inthebottomofthe acceptorplate,aflatporousfiltermaterialofpolyvinylidenefluo- ride(PVDF)islocated,inwhichanorganicsolventisimmobilized toformtheSLM.ThepHgradientarisesfrompHadjustmentofthe donorandacceptorsolution.WhenPALMEisperformedforbasic analytes,thepHinthedonorsolutionisadjustedsufficientlyhigh tomaintaintheanalytesnon-charged.Inthiswaythesolubility ofthebasiccompoundsintheaqueousdonorisreduced,thereby transferringthemintotheorganicSLM.Tofurtherpromotemass transferacrosstheSLMandintotheaqueousacceptorsolution,the pHintheacceptorisadjustedsufficientlylowtoensureionization ofthebasiccompounds.Thisisperformedtoincreasethesolubility ofthebasiccompoundsintheaqueousacceptor,andtopreventthe basiccompoundsfromback-extractingtotheorganicSLM.

Uptodate,PALMEofnon-polarbasicdrugs[13],polarbasic drugs[14],andnon-polaracidicdrugs[15]fromhumanplasma hasbeenreported,butthenumberofanalytesincludedinthese papershasbeenverylimited.Inotherwords,onlyafewPALME applicationshavebeenpublisheduptodate.However,thenumer- ous advantages associated with PALME call for expansion into newapplicationstofurtherinvestigateitspotential.The96-well formatprovideshighthroughputas it enablesextractionof 96 samplessimultaneously,efficientsampleclean-upisachievedas thesupportedliquidmembranediscriminateslargermoleculeslike proteinsandchargedcompounds,thework-flowissimple,andthe lowconsumptionoforganicsolventsrepresentsagreenchemistry approachtosamplepreparation[13,16].

In this work, PALME was investigated for the first time for thepotentialscreeningofnewpsychoactivesubstances(NPS)in plasma.ThisworkalsoinvestigatedPALMEdirectlyfromwhole bloodforthefirsttime.Theworkisapreliminarystudy,withfocus ongeneralscreening forNPSsubstances which differssubstan- tiallyintermsofmolecularproperties.Focuswasdirectedtowards optimizationofoperationalparametersandvalidation.Addition- ally,theporousPVDFfilterofthecommerciallyavailableacceptor plates,usedtosupporttheSLM,wasnotoptimalasitcausednon- specificbindingofanalytes,whichconsequentlycounteractedthe extractionperformance,aspreviouslyobserved[13].Therefore,the challengesassociatedwiththePVDFmaterialwasprioritizedearly intheoptimizationprocess.

2. Experimental 2.1. Chemicals

Methanol and sodium hydroxide were purchased from Merck Millipore (Darmstadt, Germany). Formic acid, dodecyl acetate,hexadecane,isopentylbenzene,2-nonanone,dihexylether,

MDAI was kindly donated from the National Criminal Investigation Service (NCIS/KRIPOS, Oslo, Norway). Methy- lone, pentedrone, and 2C-E were from Lipomed (Arlesheim, the Netherlands). PFA, 6-APB, and bromo-dragonfly, as well as the internal standards ¹³C₆-MDA (3,4-methyl-dioxy- amphetamine),¹³C₆-PMA(4-methoxyamphetamine),¹³C₆-MDEA (3,4-methylenedioxy-N-ethylamphetamine), and ¹³C6-PMMA (para-methoxymetamphetamine) were from Chiron AS(Trond- heim, Norway). mCPP was from Sigma-Aldrich (Steinheim, Germany), methoxetamine was from LGC (Teddington, Great Britain), and MDPV was from National Measurement Institute (Montvale,NewJersey,USA).EthylphenidateandAH-7921were fromCaymanChemicalCompany(AnnArbor,Michigan,USA).

2.2. Standardsolutions

Stocksolutionsofeachanalytedissolvedinmethanolwassup- plied by the Norwegian Instituteof Public Health (NIPH, Oslo, Norway),andstoredat−20^◦C.Thestocksolutionswereusedto prepareworkingsolutions containing allthe analytes:working solutionAwasusedintheexperimentswithPALMEfromplasma, andworkingsolutionBwasusedintheexperimentswithPALME fromwholeblood.

WorkingsolutionAcomprised10␮gmL⁻¹ ofeachanalytein deionizedwater.Thesolutionwasstoredat5^◦C,andusedtospike drug-freeplasma.Standard solutionswerepreparedbydiluting workingsolutionAwith20mMformicacid.

Forpractical reasons,workingsolutionBwasprepared with different concentrations of the analytes: MDAI (9␮gmL⁻¹), methylone (10␮gmL⁻¹), PFA (8␮gmL⁻¹), mCPP (10␮gmL⁻¹), 6-APB (9␮gmL⁻¹), pentedrone (5␮gmL⁻¹), methoxetamine (6␮gmL⁻¹),MDPV(14␮gmL⁻¹),ethylphenidate(6␮gmL⁻¹),2C- E (10␮gmL⁻¹), bromo-dragonfly (15␮gmL⁻¹), and AH-7921 (8␮gmL⁻¹).WorkingsolutionBwasstoredat−20^◦C,andwasfur- therdilutedwithdeionizedwatertopreparestandardsolutions thatwereusedtospikedrug-freewholeblood.

2.3. Internalstandards

13C6-MDA served as internal standard for all the analytes in the PALME experiments from plasma. In the PALME exper- iments from whole blood, the analytes were allocated one of thefollowinginternal standards:¹³C6-MDA(pentedrone), ¹³C6- PMA(MDAI,methylone,PFA,mCPP,6-APB,methoxetamine,and ethylphenidate),¹³C₆-2C-B(2C-E),¹³C₆-MDEA(MDPVandbromo- dragonfly), and ¹³C6-PMMA (AH-7921). The internal standards weredissolvedindeionizedwaterandstoredat4^◦C.

2.4. Biologicalmatrices

Humanwholebloodwithsodiumfluorideandheparinasaddi- tives was supplied by the Blood Bank of Oslo, Oslo University Hospital,Norway.Plasmawasstoredinplastictubesat−25^◦C,and wholebloodwasstoredinbrownglassbottlesat−20^◦C.Thebio- logicalmatriceswerethawedpriortopreparationofthebiological samples.

2.5. PALMEprocedure

PALMEwasperformedusinga96-welldonorplateofpolypropy- lenewith0.5mLwellsfromAgilent(Santa Clara,CA,USA),and a96-wellacceptorplatefromMillipore(Billerica,MA,USA)with

Fig.1. PictureofcommerciallyavailableequipmentusedforPALME(1),depictingthedonorplate(A),theacceptorplate(turnedupsidedown)withPVDFmaterial(B),and thetoplid(C).SchematicillustrationofaPALMEextractionunit(2).

polyvinylidenefluoride(PVDF)assupportfortheliquidmembrane (SLM).TheporesizeofthePVDFmaterialwas0.45␮mandthe internaldiameter6.0mm.Alidwasusedtopreventevaporationof theacceptorsolutionsduringPALME.

When performing PALME from plasma, sample aliquots of 125␮Lwerepipettedintothedonorwellsinthedonorplate.This waseitherblankplasmaorplasmaspikedwiththeanalytes.To alkalizethesamples,115␮Lof40mMNaOHwasaddedtothesam- ple.Finally,10␮Lofinternalstandardwasadded,makingafinal samplevolumeof250␮L.

WhenperformingPALMEfromwholeblood,samplealiquots of150␮Lwerepipettedintothedonorwellsinthedonorplate.

Thiswaseither100␮Lwholebloodspikedwith50␮Lstandard containingtheanalytes,or100␮Lwholebloodand50␮Ldeionized water(blankblood).Toalkalizethesamples,75␮Lof80mMNaOH wasadded.Finally,25␮Lofinternalstandardwereadded,making afinalsamplevolumeof250␮L.

TopreparetheSLM,5␮Ldodecylacetatewith1%trioctylamine (w/w)waspipettedontothePVDFmaterialintheacceptorplate.

Finally,50␮Lofacceptorsolutioncomprising20mMformicacid wasaddedtothewellsintheacceptorplate.Thedonorandacceptor platewasclampedtogether,andthewholeset-upwasplacedona platformshaker(Vibramax100,HeidolphInstruments,Schwabach, Germany),providingagitationof900rpmtopromotetheextrac- tion.PALMEwascarriedoutfor120min.

2.6. UHPLC–MS/MSanalysisofplasmasamples

UHPLC–MS/MS analysis of PALME extracts obtained from plasmasampleswasperformedwithachromatographic system consistingofaDionexUltiMate3000RSPump,ColumnCompart- ment,andAutosampler,andaThermoScientificLTQXLLinearIon TrapMassSpectrometerfromThermoScientific(SanJose,CA,USA).

ChromatographicseparationwasobtainedwithanAcquityUPLC^® HSST3columnfromWaters(Wexford,Ireland)withalengthof 100mm,aninnerdiameterof2.1mm,a particlesizeof1.8␮m, andaporesizeof100Å.Thecolumntemperaturewas40^◦C.Data acquisitionandprocessingwasperformedusingXcaliburversion 2.2SPI48softwarefromThermoScientific.

Mobile phase A and B comprised 20mM formic acid and methanol in the ratios 95:5 (v/v) and 5:95 (v/v), respectively.

Throughouttheanalysis,themobilephases werepumpedwith anoverallflowrateof0.3mLmin⁻¹followingapre-setgradient:

15%Bat0.00min,50%Bat15min,100%Bat15.1min,100%Bat 16.9min,15%Bat17min,and15%Bat20min.Totalruntimewas 20min,andtheinjectionvolumewas2.0␮L.Ionizationwasper- formedwithelectrosprayionization(ESI)inthepositiveionization mode(ESI+)withavoltageof5kV.Analysiswasperformedusing selectedreactionmonitoring(SRM).Thetransitionsandcollision energiesareshowninTable1.

Table1

MSparametersforLC–MS/MSanalysisofPALMEextractsfromplasmasamples.

SRMtransitions

Compound Precursor Fragment Collisionenergy

(%)

MDAI 178.1 161 33

Methylone 208.1 190 34

PFA 154.2 137 18

mCPP 197.2 119 27

6-APB 176.1 159 39

Pentedrone 192.2 174 32

Methoxetamine 248.1 203 49

MDPV 276.3 205 50

Ethylphenidate 248.2 84.1 35

2C-E 210.2 178.1 45

Bromo-dragonfly 294.2 277 31

AH-7921 329.2 190.1 31

13C6-MDA 186.1 169.1 35

2.7. UHPLC–MS/MSanalysisofwholebloodsamples

UHPLC–MS/MSanalysisofPALMEextractsobtainedfromwhole blood sampleswasperformedusinganintegrated systemfrom Waters(Milford,MA,USA)withanAcquity^TMUltraPerformanceLC.

Chromatographicseparationwasperformedat65^◦ConanAcquity UPLC^®HSST3columnwithalengthof100mm,aninnerdiameter of2.1mm,aparticlesizeof1.8␮m,andaporesizeof100Å.Data acquisitionwasperformedusingMassLynx4.1software(Waters Corp.,Milford,MA,USA),anddatawereprocessedwiththeTarget- Lynxquantificationprogram(WatersCorp.).

Mobile phase A and B consisted of 10mM ammonium for- mate (pH 3.1) and methanol, respectively. The flow rate was 0.5mLmin⁻¹,andtheanalysiswasperformedwiththefollowing gradient:2.5%Bat0.00min,35%Bat6.75min,65%Bat8.00min, 100%Bat8.01min,100%Bat9.00min,and 2.5%Bat9.01min.

Totalruntimewas11min,andtheinjectionvolumewas3.0␮L.MS detectionwasperformedonaWatersQuattroPremierXEtriple- quadrupoleMS,(WatersCorp.,Milford,MA,USA).Ionizationwas performedwithelectrosprayionizationinthepositivemodewith avoltageof1kV.Analysiswasperformedusingselectedreaction monitoring.TheSRMtransitions andcollision energiesarepre- sentedinTable2.

2.8. MethodvalidationofPALMEfromplasma

Thefollowingparametersweretakenintoconsideration:lin- earity,limitsofquantification(LOQ),intra-andinter-dayprecision, accuracy,extractionrecoveries,carry-over,andmatrixeffects.

Thelinearityof themethodwasstudiedforeach analyteon eightconcentrationlevels(n=4)rangingfrom5to1000ngmL⁻¹. Weighted quadratic calibration curves (1/x) were constructed by the computer software Xcalibur. The LOQ for each analyte

Compound Precursor Fragment1 Fragment2 Collisionenergy (%)

MDAI 178.1 161 131 18

Methylone 208.1 190.1 160 20

PFA 154.2 137.2 109 10

mCPP 197.2 154.1 119.1 30

6-APB 176.1 159.1 131 14

Pentedrone 192.2 174.1 132 18

Methoxetamine 248.1 203.1 121 15

MDPV 276.3 175.1 126.1 28

Ethylphenidate 248.2 84.1 56 28

2C-E 210.2 193.1 178.1 20

Bromo-dragonfly 294.2 198.1 141.1 30

AH-7921 329.2 284.2 190.1 28

wasdeterminedfromscalar dilutionsoftheanalytesin plasma (0.5–1000ngmL⁻¹).TheLOQwastheconcentrationvaluegiving anS/N=10.Intra-dayprecisionandaccuracywasstudiedatthree concentrationlevelsand withtenreplicates,whereas inter-day precisionwasdeterminedover three consecutivedays at three concentrationlevelsandwithfourreplicates.

The extractionrecoveries were determined on two concen- trationlevels.Eightreplicatesofblankplasmawerespikedwith analytesbeforePALME andcompared tofourreplicatesofpost extractionspikedsamples.Theextractsfromtheplasmasamples thatwerespikedafterPALMErepresented100%recoveryandwere usedtodeterminetheextractionrecoveriesfromtheplasmasam- plesthatwerespikedbeforePALME.

Potentialcarry-overintheUHPLC–MS/MSwasinvestigatedby injectinga tentimesmore concentratedsample thanthehigh- eststandardof1000ngmL⁻¹,followedbytheinjectionofthree blankplasmasamples.AfterUHPLC–MS/MS,thechromatograms obtainedfromthe blankplasma sampleswere investigated for potentialchromatographicpeaks,servingasproofsofcarry-over.

Finally,toevaluateanypotentialionsuppressionorenhance- ment(matrixeffects)duetotheplasmamatrix,acceptorsolutions fromextractedblankplasma(spikedwithanalytesafterPALME, n=2)werecompared withuntreated acceptorsolutions(spiked withanalyteswithout performingPALME, n=2). Thiswas per- formedfortwoconcentrationlevels,andthematrixeffectswere quantifiedasdescribedinsection2.10.(Calculations).

2.9. MethodvalidationofPALMEfromwholeblood

Methodvalidationfromwholebloodwascarriedoutsimilarly tothevalidationfromplasma.DetailsaregiveninTable6.

2.10. Calculations

Recovery(R%)wascalculatedaccordingtothefollowingequa- tionforeachanalyte:

R%= nafinal

n_dinitial×100%= Va

V_d× Cafinal C_dinitial×100%

wheren_distheinitialnumberofanalytemolespresentinthedonor solution(sample),andnaisthefinalnumberofanalytemolescol- lectedintheacceptorsolution.V_dandVarepresentsthedonorand acceptorvolume,respectively,whereasC_dinitialandC_afinalrep- resentstheinitialandfinalanalyteconcentrationinthedonorand acceptorsolution.

MDAI C10H11NO2 10.0 1.1 177.2

Methylone C11H13NO3 8.0 1.2 207.2

PFA C9H12FN 10.0 2.0 153.2

mCPP C10H13ClN2 8.9 2.2 196.7

6-APB C11H13NO 10.0 2.0 175.2

Pentedrone C12H17NO 8.2 2.6 191.3

Methoxetamine C15H21NO2 8.1 2.9 247.3

MDPV C16H21NO3 7.3 3.0 275.3

Ethylphenidate C15H21NO2 9.1 2.6 247.3

2C-E C12H19NO2 9.7 2.2 209.3

Bromo-dragonfly C13H12BrNO2 9.8 2.9 294.1 AH-7921 C16H22Cl2N2O 9.5 4.0 329.3

Matrixeffects(ME)werecalculatedaccordingtoMatuszewski etal.[17]asfollows:

ME%=B A×100%

whereBisthepeakareaobtainedfromspikinganextractacquired afterextractionfromblankwholebloodorplasma.,andAisthe peakareaobtainedfromaneatstandardsolution.

3. Resultsanddiscussion

Parallelartificialliquidmembraneextraction(PALME)wasfor thefirsttime performedfornewpsychoactivesubstances(NPS) inplasmaandwholeblood.Optimizationofoperationalparam- eters was performed before the final methods were validated.

TwelvedifferentNPSwereselectedasmodelanalytes,andwere allhydrophobicbases,coveringabroadlogPrangefrom1.1to4.0 (seeTable3forexactlogPvalues,molecularformulas,pKavalues, andmolecularweights).

PALMEwasperformedwithcommerciallyavailableequipment.

Thiswasdefinitelyanadvantage,butthePVDFfilterinthebottom oftheacceptorplateswasnotoptimal,basedonpreviousobser- vations[13].Non-specificbindingofanalytestothePVDFmaterial sacrificedthelinearityastheextractionrecoveriesdecreasedwith decreasinganalyteconcentration.Polypropyleneisanalternative filtermaterialusedinHF-LPME[18],andinthefirstPALMEpaper thePVDFmaterialwasreplacedwithpolypropylene.Thisimproved theextractionperformance,but96-wellplateswithpolypropylene filterswithappropriatedimensionsarenotyetcommerciallyavail- able.OptimizationofPALMEperformancewithPVDFfilterswas thereforeinvestigatedindetail.

3.1. Optimizationofoperationalparameters(PALMEfrom plasma)

Differentoperationalparameterswerestudiedandoptimized.

First, different organicsolvents were tested as potential liquid membranes.Sixpuresolventswereselectedbasedonearlierexpe- rienceswithHF-LPMEandPALME[13,19].Inallcases,5␮Lsolvent waspipettedontothePVDFmaterialandwasimmobilizedbycap- illaryforces,withoutfurtheroptimizationoftheSLMvolume.The solventswerenon-volatileandimmisciblewithwatertoprevent partialevaporationduringPALME,andtoavoidleakageoftheSLM tothedonor/acceptorsolution.Extractionrecoveries(n=4)after 30minofPALMEarepresentedinTable4.Hexadecanewasineffi- cient,probablybecauseitwastoohydrophobic.Isopentylbenzene, dodecylacetate,and2-nonanoneprovidedthehighestrecoveries.

Isopentylbenzenewasnottestedfurtherforboilingpointreasons (195^◦C),and2-nonanonewasnotselectedduetowatersolubil- ity(1.1g/L)[20].Thus, dodecylacetate(watersolubility0.02g/L,

Table4

RecoverydataforPALMEfromplasmawithdifferentorganicsolventsservingasSLM.

%Recovery(%RSD),n=4

Compound Dihexylether 2-nonanone Dodecylacetate Isopentyl-benzene 2-nitrophenyloctylether Hexadecane

MDAI 16(5) 57(3) 20(8) 51(13) 10(14) 1(26)

Methylone 51(7) 87(3) 64(2) 89(2) 39(4) 8(11)

PFA 73(2) 83(3) 69(4) 93(5) 45(10) 35(10)

mCPP 87(4) 90(4) 90(4) 102(2) 76(6) 35(6)

6-APB 75(5) 73(3) 79(5) 97(4) 57(4) 26(10)

Pentedrone 101(3) 100(3) 110(2) 110(4) 101(3) 94(2)

Methoxetamine 104(7) 104(5) 110(2) 112(2) 103(3) 83(6)

MDPV 88(11) 57(5) 97(6) 92(14) 99(6) 65(12)

Ethylphenidate 97(2) 89(7) 102(4) 103(5) 94(6) 66(7)

2-CE 17(8) 44(7) 28(4) 50(8) 23(9) 1(20)

Bromo-dragonfly 53(7) 17(10) 67(7) 74(12) 67(9) 23(12)

AH-7921 65(14) 28(5) 79(8) 72(11) 85(3) 30(16)

Meanvalues 60(6) 68(6) 68(6) 75(12) 58(10) 33(14)

Fig.2.Theeffectoftrioctylamine(TOA)addedtothesupportedliquidmem- brane.ExtractionperformancefromPALMEexperimentsfromplasmawith(A) SLM=dodecylacetate,and(B)SLM=dodecylacetate+1%TOA.

boilingpoint265^◦C)wasselectedfortheremainingexperiments [20].

Asseen fromTable4, acceptableextractionrecoverieswere obtainedforalltheanalytes.Still,insomeinitialexperiments,sig- nificantnon-specificbindingtothePVDFmaterialwasobserved.

Particularly,recoveriesweretosomeextentimprovedbyincreas- ing the total load of NPS in the samples. To circumvent this, trioctylamine(TOA)wasaddedtotheorganicsolventservingas SLM(dodecylacetate)tomaskthePVDFbindingsites.Toevaluate theeffectofTOAintheliquidmembrane,PALMEwasperformed bothwithandwithouttheadditionof1%TOA(w/w).The1%con- centrationofTOAwasselectedwithoutfurtheroptimization,based onearlierexperienceswithrelatedsubstances(unpublishedwork).

ThepercentageadditionofTOAtotheorganicsolventwasnotfur- theroptimizedinthiswork.TheresultsareshowninFig.2fora representativeselectionofeightanalytes(n=4).Thefigureshows thattheeffectofTOAwasanalyte dependent.Forexample,the effectofTOAwasappreciableforthecompoundAH-7921,whereas formethoxetaminetheeffectwaslesssignificant.Still,additionof 1%TOAtotheSLMwasconsideredbeneficialformostanalytes,and theoverall extractionperformance wasimprovedsubstantially.

Dodecylacetatewith1%TOAwasthereforechosenasoptimalSLM forthecontinuedwork.

In a final optimizationexperiment, the extractiontime was addressed.PALMEwasperformedfromplasma,and theextrac- tionrecoveriesweremeasuredafter5min,20min,45min,60min, 90min,120min,and180min(n=4).Fig.3showstheextraction recoveriesforpentedrone, methylone,and2C-E.Theseanalytes were selected to illustrate three kinetic patterns: some ana- lytes reached equilibrium within 45min (pentedrone, MDPV,

AH-7921,mCPP,methoxetamine,andethylphenidate),someana- lytescontinuedtoincreaseevenafter45minofPALME(methylone, bromo-dragonfly,PFA,and6-APB),andfinally,someanalyteswere extractedwithveryslowkinetics(2C-EandMDAI)withnoequi- libriumestablished,evenafterthreehoursofPALME.Theanalytes withrapidkineticsweremostlywithlogP>2.5,butkineticswas notonlyrelatedtologP.Otherchemicaldescriptorswerealsocon- sidered,suchaspolarsurfacearea,numberofhydrogenbinding donorandacceptorsites,andnumberofrotationalbonds,butthe currentdatasetwasinsufficienttodevelopaclearrelationbetween extractionkineticsandmolecularproperties.Thelattercompounds wererelativelypolarsubstances(lowlogP).Thefinalextraction timewassetto120minasacompromisebetweenthroughputand recovery.

3.2. ResultsfromthemethodvalidationofPALMEfromplasma SuccessfulPALME fromplasma wasachieved froma 250␮L alkalizeddonorsolutioncomprisingtheplasmasample(125␮L), theinternalstandard(10␮L),and40mMNaOH(115␮L).Theana- lyteswereextractedacrossanSLMof5␮Ldodecylacetatewith1%

TOA(w/w),andfinallyintoanacidicacceptorsolutionof20mM formicacid[13].PALMEwasperformedfor120min,andtheextrac- tionwaspromotedwithagitationat900rpm[13].ThefinalPALME methodwasvalidatedforthetwelveNPSpresentedinTable3,and with¹³CMDAasinternalstandard.Thevalidationwasperformed accordingtoFDAguidelines,andtheresultsaresummarizedin Table5.

Thecalibrationcurvesshowedthatthemethodwaslinearin therangefrom5to1000ngmL⁻¹,andquadraticcorrelationcoef- ficients>0.990wereobtainedforalltheanalytes,exceptforMDAI (r²=0,988). Table5 shows that theLOQswere 0.78ngmL⁻¹ or lowerforalltheanalytes,andthisconfirmedthatquantification wasfeasibledowntothe5ngmL⁻¹pointofthecalibrationcurve.

Intra-andinter-dayprecisionwasalsosatisfactory,andwithinthe range4–13%RSDand5–20%RSD,respectively.Theaccuracywas within±20%.

Extractionrecoveries weredeterminedat two concentration levelsto investigatewhetherthe recoverymeasurements were affectedbyanalyte concentration.Therecoverydata inTable5 shows that the NPS with log P>2 obtained a higher recovery compared totheNPS withlogP<2. Still, alltheanalytes were extractedwithhighprecision,provingthattheNPSweresuccess- fullyextractedfromplasma.

ItwasobservednochromatographicpeaksintheLC–MS/MS chromatogramsfromtheblankplasmasamplesthatwereinjected afterthesamplewithhighconcentrationofanalytes,andtherefore itwasconcludedthatcarry-overwillnotbelikelytoaffecttheana-

Fig.3. Extractionrecoveriesfromplasmaversusextractiontime(5–180min)witherrorbarsrepresenting±1standarderror(SE).

Table5

ValidationdataforthePALMEmethodfromplasma.

Compound Concentration (ngmL⁻¹)

Intra-day precision^a,RSD (%)

Inter-day precision^b,RSD (%)

Accuracy^c(%) Recovery^d(%) LLOQ(ngmL⁻¹) Calibrationrange (ngmL⁻¹)

Linearity(r²)

MDAI 7.5 11 9 20 26 0.7 5–1000 0.988

75 8 13 11

750 13 14 6 25

Methylone 7.5 9 12 −17 80 0.1 5–1000 0.992

75 7 11 −3

750 8 13 −4 95

PFA 7.5 7 9 −15 94 0.5 5–1000 0.996

75 4 10 −4

750 9 14 −7 91

mCPP 7.5 5 5 −12 94 0.8 5–1000 0.994

75 6 10 5

750 6 20 −2 104

6-APB 7.5 4 7 −10 86 0.5 5–1000 0.994

75 5 10 5

750 8 11 −1 96

Pentedrone 7.5 6 6 −15 109 0.4 5–1000 0.992

75 5 11 6

750 9 11 −2 117

Methoxetamine 7.5 8 5 −6 98 0.3 5–1000 0.993

75 6 11 −7

750 9 10 −4 101

MDPV 7.5 7 8 −10 100 0.1 5–1000 0.995

75 7 11 −6

750 11 12 −8 99

Ethylphenidate 7.5 5 7 −13 105 0.3 5–1000 0.999

75 6 12 3

750 8 16 −1 109

2C-E 7.5 5 9 −6 44 0.4 5–1000 0.992

75 5 10 0

750 6 10 −4 52

Bromo-dragonfly 7.5 5 5 −11 95 0.5 5–1000 0.997

75 4 10 5

750 6 10 −2 107

AH-7921 7.5 7 11 −6 101 0.2 5–1000 0.997

75 8 13 −2

750 9 11 2 105

aIntra-dayprecisionwasmeasuredatthreeconcentrationlevels(n=10).

b Inter-dayprecisionwasmeasuredatthreeconcentrationlevelsatthreeconsecutivedays(n=4).

c Accuracywasmeasuredfromintra-dayprecisionatthreeconcentrationlevels(n=10).

d Recoverywasmeasuredattwoconcentrationlevels(n=8).

Table6

ValidationdataforthePALMEmethodfromwholeblood.

Compound Concentration (ngmL⁻¹)

Intra-day precision^a,RSD (%)

Inter-day precision^b,RSD (%)

Accuracy^c(%) Recovery^d(%) LLOQ(ngmL⁻¹) Calibrationrange (ngmL⁻¹)

Linearity(r²)

MDAI 89 6 11 −3 11 44 44–266 0.997

Methylone 104 13 12 −8 31 52 52–311 0.990

PFA 77 8 7 −3 40 38 38–230 0.992

mCPP 98 9 8 −6 66 98 98–295 0.993

6-APB 88 5 6 −8 38 44 44–263 0.993

Pentedrone 48 8 9 −2 83 25 25–143 0.992

Methoxetamine 62 8 9 −6 86 62 62–185 0.990

MDPV 138 7 11 8 54 138 138–413 0.997

Ethylphenidate 62 10 7 1 63 62 62–185 0.989

2C-E 105 4 9 −9 3 105 105–314 0.996

Bromo-dragonfly 147 19 19 9 38 147 147–441 0.985

AH-7921 82 18 13 −3 32 82 82–247 0.970

aIntra-dayprecisionwasmeasuredatoneconcentrationlevel(n=10).

bInter-dayprecisionwasmeasuredatoneconcentrationlevelatsevenconsecutivedays(n=2).

c Accuracywasmeasuredfrominter-dayprecisionatoneconcentrationlevel(n=2).

d Recoverywasmeasuredatoneconcentrationlevel(n=10).

lyticalresultswithexpectableconcentrationsinbiologicalsamples.

Thequantifiedmatrixeffectsweresatisfactoryandwithin±15%.

3.3. ExperimentswithPALMEfromwholeblood

This was the first time PALME was performed from whole blood.For simplicity,wepreferredtousethesameexperimen- talconditionsasfortheplasmasamples,butduetocompositional differences,thepHconditioninthewholebloodsampleswaspar- ticularlyaddressed.AroughinspectionofthesamplepH,usingpH paper,revealedthattheinitialsamplecompositionfailedtoalkalize thebloodsamplessufficiently.Thiswasduetothebuffercapacity ofwholeblood,providingastablepHof7.4.AtthispH,someofthe analyteswiththehighestpKavalueswereprotonatedandconse- quentlypreventedfromenteringtheorganicSLM.Toensurethat allthebasicanalyteswereunchargedinthedonorsolution,the sampleswerepreparedasdescribedintheexperimentalsection, butwithdifferentconcentrationsofNaOH.Theaimwastoachieve afinalsamplepHof12andinvestigatehowtheelevatedsample pHaffectedtheextractionperformance,usingextractionrecov- eryassuccessparameter.NaOHconcentrationsof10mM,20mM, 30mM,40mM,60mM,80mM,100mMwastested(n=4,datanot shown).Thehighestrecoverieswereobtainedwith75␮L80mM NaOHaddedtothesample,resultinginafinalconcentrationof 24mMNaOHinafinaldonorsolutionof250␮L.

To investigate whether foaming and potential carry-over occurredbetweenthedonorwellsinthedonorplateduringPALME, anentire96-wellplatewasfilledwithbloodsamples.Everyother donor wellwas filled withblank samples (blank wholeblood) andbloodsamplesspikedwithanalytes,respectively.Notraces ofanalytesweredetectedintheblanksamplesafterPALME,and thissupportedtheabsenceofcarry-overandcrosscontamination betweenindividualdonorwells.

3.4. ResultsfromthemethodvalidationofPALMEfromwhole blood

PALMEofthetwelveNPS(Table3)fromwholebloodwasper- formedfrom a 250␮L alkalized donor solutioncomprising the bloodsample(100␮L),deionizedwater(50␮L),internalstandard (25␮L),and 80mM NaOH(75␮L).Theanalyteswereextracted acrossanSLMof 5␮L dodecylacetatewith1%TOA(w/w),and finallyintoanacidicacceptorsolutionof20mMformicacid[13].

PALMEwasperformedfor120min,andtheextractionwaspro- motedbyagitationat900rpm[13].ThefinalPALMEmethodwas validated,andtheresultsaresummarizedinTable6.

ThemethodwaslinearintherangefromtheLOQofeachanalyte tothehighestcalibrationlevel.Correlationcoefficients>0.990were obtainedforall theanalytes,except forethylphenidate (0.988), bromo-dragonfly (0.985), and AH-7921 (0.970). Table 6 shows theLOQsforeachanalyte,andtheywereallconsideredsatisfac- tory.Intra-dayprecisionwaswithin±20%forMDAI,mCPP,6-APB, pentedrone,methoxetamine,MDPV,2C-E,andbromo-dragonfly, whereasinter-dayprecisionwaswithin±20%foralltheanalytes.

Accuracies were within±9%and werein compliance with the requirementsforbioanalyticalmethodvalidation(<15%).

No chromatographic peakswere observed inthe LC–MS/MS chromatogramsfromtheblankbloodinthecarry-overexperiment, orintheLC–MS/MSchromatogramsfromthespecificityexperi- ment.Therefore,itwasconcludedthatthemethodwasspecific and provided nocarry-over. Thequantifiedmatrix effects were satisfactoryandwithin±15%.

4. Conclusion

Inthiswork,thepotentialofparallelartificialliquidmembrane extraction (PALME) of new psychoactive substances (NPS) was demonstratedwithcommerciallyavailableequipmentforthefirst time.TwelvecompoundsrepresentingaselectionofNPSweresuc- cessfullyextractedfromplasmaandwholeblood.Basedoninitial optimizationexperimentsandpreviousexperiences,twoseparate PALMEmethodsweredevelopedforplasmaandwholeblood.The finalmethodswerevalidated,andtheresultswereinaccordance toFDAguidelines.

ThisisthefirstPALMEapplicationdevelopedforforensicanal- ysis.Extensivesample clean-up,possibilityfor highthroughput, anda simplework-flowareadvantages associatedwithPALME.

Theaqueousextractsfrom PALMEaredirectlycompatible with LC–MS,andnoevaporationandreconstitutionisrequired.PALME canbeperformedwithcommerciallyavailableplates,andonly3␮L organicsolventisusedpersample.Thetotalconsumptionforpro- cessing96samplesisthereforelessthan0.3mL,andthisrepresents averyinterestingapproachtogreensamplepreparation.Basedon thesegeneraladvantages,andtheapplicationsdevelopedinthis work,PALMEisexpectedtobeavaluabletoolforforensiclabora- toriesinthenearfuture.

Acknowledgement

TheResearchCouncilofNorwayisacknowledgedforfinancial supportthroughGrantno.231917.

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