Technicalnoteno.AS182

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FullyAutomatedMethodusingDispersiveLiquidLiquidMicroExtraction(DLLME)forExtractableandLeachablestudies

AlanHutchinson,GlaxoSmithKline.,Stevenage,Hertfordshire(UK)DanCarrier,AnatuneLtd.,Girton,Cambridgeshire

Introduction

Dispersive Liquid Liquid Micro Extraction (DLLME) is a technique used toextractanalytesfromaproductmatrixandprovidealevelofenrichmentfortraceanalysistesting.

Adispersiveagentisaddedtothesamplepriortotheadditionofachlorinatedsolvent, either dichloromethane or chloroform. For the work with GSKchloroformwasused.

Thedispersiveagentallows thechlorinated solventonaddition to formanemulsion thus creating a huge surface area for the chlorinated solvent toextracttheanalytesofinterest.Afterthesolutionismixedforaperiodoftime,thesampleiscentrifugedandthebottomlayerisinjectedontotheGC.

Manual DLLME can become time consuming and require large volumes ofsampleandsolvents.Theuseofarobustfullyautomatedmethodallowsananalyst to perform other laboratory tasks and increases throughput withovernightanalysis.

AnatuneandGSKhaverecentlydevelopedacompletelyautomatedsolution,usingfastagitationandcentrifugation.Figure1showsinstrumentationwhichcancarryoutthissolution.

Figure1-DualHeadMPSxtwithCentrifuge(CF-200)andVortex(mVorx)onaGC/Q-TOF

A video is enclosed below which shows the addition (in slowmotion) ofdichloromethanetoasolutioncontainingPropan-2-olandwater. Itcanbeseenhowquicklythedichloromethaneisbrokenupintoanemulsion.

EnclosedVideo

Figure2showsthesolutionafterithasbeencentrifugedat4500rpm.Thevialisahighrecoveryvialenablingwithdrawalofalowvolume(typicallylessthan50µL)ofthechlorinatedsolventfromthebottomofthevial.

Figure2–High recoveryvialwith lowvolumeof thechlorinatedsolventpresentatthebottomofthevial(GSKuseChloroform).

Instrumentation

DualHeadGERSTELMPSGERSTELmVORXAgilentGC/Q-TOF7200AnatuneCF-200Centrifuge

Method

2mLofabiopharmaceuticaldrugproductsamplewastransferredmanuallytoa10mLvial.Threedifferentstocksolutionscontainingtheanalyteslistedin Table 4 were prepared in Propan-2-ol at the levels stated in Table 1.Additionally,aninternalstandardofFluorened10wasaddedtoeachtestmixlevel at a concentration of 13.5 µg/mL, so equivalent to 0.33 µg/mL persample. 2,4-ditertbutylphenol was in the test mix at five times theconcentrationtoallowforintrinsicquantitiesfoundinthedrugproduct.

Level SpikeConc[µg/mL] FinalConcentraioninsample[µg/mL]

1 13.5 0.332 68.0 1.663 136.5 33.3

Table1Testmixturespikinglevels

Dichloromethaneaftercentrifugation

Technicalnoteno.AS182

Afteradditionofthetestmixturetothebiopharmaceuticaldrugproduct,4.5mL of Propan-2-ol was added to a 10 mL vial to crash out thebiopharmaceuticalprotein.ThevialwasthentransportedtothemVORXandvortexedat3000rpmfor30seconds.Thevialwasthencentrifugedat4000rpmfor10minutes.Upto6vialscanbecentrifugedatonetime.

After centrifugation, 1.5 mL of the extract was added to a 10 mL Highrecoveryvial.75µLofChloroformwasthenaddedtothehighrecoveryvialandvortexedfor30secondsat3000rpm.4.0mLofwaterwasaddedasadisperserandvortexedagainfor30secondsat3000rpm.Thesolutionwasthencentrifugedat4500rpmfor5minutes.FromtheChloroformsolventlayer35µLwasaddedtoahighrecoveryGCvialreadyforinjectionontotheGC/Q-TOF

Results

Precisionwascarriedoutonsixsamplesspikedat0.33µg/mLwhichresultedin10%RSDorlessforallcomponentsofthetestmix.

Analyte %RSD

4-MethylBenzaldehyde 1.7Dodecane 3.8

1,6-Dibromohexane 2.8Tetradecane 5.0

4-Pentylphenol 1.92,4-Ditertbutylphenol 5.3

ButylatedHydroxytoluene 1.61-Bromododecane 10.2

3,5-di-Tert-Butyl-4-hydroxybenzaldehyde 2.2Octadecane 1.8

1-Bromopentadecane 2.3

Table2–%RSDfor6differentextractionsat0.33µg/mL

Recoveryexperimentswerecarriedoutatthethreeconcentrationlevelsandthreepreparationsofeach.Thesewerecarriedouttoensuretherewerenomatrixeffectsfromthebiopharmaceuticalproduct.

Analyte%Recovery

0.33µg/mL

1.66µg/mL

33.3µg/mL

4-MethylBenzaldehyde 106.7 104.3 99.2Dodecane 108.0 108.9 102.01,6-dibromohexane 101.2 100.6 101.4Tetradecane 111.6 107.6 102.94-pentylphenol 98.0 104.1 101.72,4-ditertbutylphenol 132.1 102.5 92.6ButylatedHydroxytoluene 102.8 108.2 101.01-bromododecane 114.1 102.9 101.93,5-ditertbutyl-4-hydroxybenzaldehyde

100.5 100.5 101.2

Octadecane 101.1 102.4 102.11-bromopentadecane 99.7 97.8 101.1

Table3.Recoveryexperimentsperformedatthreedifferentconcentrations

Analyte r24-MethylBenzaldehyde 0.995Dodecane 0.9981,6-dibromohexane 0.984Tetradecane 0.9974-pentylphenol 0.9912,4-ditertbutylphenol 0.911ButylatedHydroxytoluene 0.9791-bromododecane 0.9953,5-ditertbutyl-4-hydroxybenzaldehyde 0.998Octadecane 0.9971-bromopentadecane 0.998

Table4.r2valuesforthelinearityexperiments

Figure3showsalinearityplotfor3,5-Di-Tert-Butyl-4-Hydroxybenzaldehyde

Figure3.linearityplotfor3,5-Di-Tert-Butyl-4-Hydroxybenzaldehyde

Figure4showsatypicalchromatogramofasamplespikedwithtestmixatthe0.33µg/mLlevel.

Figure4.ChromatogramofSampleat0.33µg/mLlevel

Discussion

ItisnowpossibletofullyautomateDispersiveLiquidLiquidMicroExtractionusingthehighrecoveryvialswithautomatedmixingandcentrifugation.Thiswill increase throughput within GSK over the manual method. Please letAnatuneknowifyouneedanyfurtherinformation.