Optimisation of pressurized liquid extraction using a multivariate chemometric approach for the determination of anticancer drugs in sludge by ultra high performance liquid chromatography–tandem mass spectrometry

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: DOI:10.1016/j.chroma.2013.01.114

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To cite this version:

Seira, Jordan and Claparols, Catherine and Joannis-Cassan, Claire and

Albasi, Claire and Montréjaud-Vignoles, Mireille and Sablayrolles,

Caroline Optimisation of pressurized liquid extraction using a multivariate

chemometric approach for the determination of anticancer drugs in sludge

by ultra high performance liquid chromatography–tandem mass

spectrometry. (2013) Journal of Chromatography A, vol. 1283 . pp. 27-38.

ISSN 0021-9673

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Optimization

of

pressurized

liquid

extraction

using

a

multivariate

chemometric

approach

for

the

determination

of

anticancer

drugs

in

sludge

by

ultra

high

performance

liquid

chromatography–tandem

mass

spectrometry

Jordan

Seira

_,

_Catherine

_Claparols

_,

_Claire

_{Joannis-Cassan}

_,

_Claire

_Albasi

_,

Mireille

Montréjaud-Vignoles

_,

_Caroline

_Sablayrolles

a_{UniversitédeToulouse,INP-ENSIACET,CNRS,LaboratoiredeGénieChimique,4alléeEmileMonso,F-31432ToulouseCedex4,France} b_{UniversitédeToulouse,INP-ENSIACET,INRA,LaboratoiredeChimieAgro-Industrielle,4alléeEmileMonso,F-31432ToulouseCedex4,France} c_{CNRS,LaboratoiredeChimiedeCoordination,205routedeNarbonne,BP44099,F-31077ToulouseCedex4,France}

d_{UniversitédeToulouse,UPS,ServiceCommundeSpectrométriedeMasse,118routedeNarbonne,F-31077ToulouseCedex4,France}

Keywords: Anticancerdrugs Sludge

Experimentaldesign Pressurizedliquidextraction Ultrahighperformanceliquid chromatography

Tandemmassspectrometry

a

b

s

t

r

a

c

t

Thepresentpaperdescribesananalyticalmethodforthedeterminationof2widelyadministered anti-cancerdrugs,ifosfamideandcyclophosphamide,containedinsewagesludge.Themethodreliesonthe extractionfromthesolidmatrixbypressurizedliquidextraction,samplepurificationbysolid-phase extractionandanalysisbyultrahighperformanceliquidchromatographycoupledwithtandemmass spectrometry.Thedifferentparametersaffectingtheextractionefficiency wereoptimizedusingan experimentaldesign.Solventnaturewasthemostdecisivefactorfortheextractionbutinteractions betweensomeparametersalsoappearedveryinfluent.Themethodwasappliedtosevendifferenttypes ofsludgeforvalidation.Theperformancesoftheanalyticalmethoddisplayedhighvariabilitybetween sludgeswithlimitsofdetectionspanningmorethanoneorderofmagnitudeandconfirmingthe rele-vanceofmulti-samplevalidation.Matrixeffecthasbeendeterminedasthemostlimitinganalyticalstep forquantificationwithdifferentextentdependingonanalyteandsludgenature.Foreachanalyte,the useofdeuteratedstandardspikedattheverybeginningensuredthecompletecompensationoflosses regardlessofthesamplenature.Thesuitabilityofthemethodbetweenfreshlyspikedandaged sam-pleshasalsobeenverified.Theoptimizedmethodwasappliedtodifferentsludgesamplestodetermine theenvironmentallevelsofanticancerdrugs.Thecompoundsweredetectedinsomesamplesreaching 42.5mg/kgDMinifosfamideforthemostcontaminatedsample.

1. Introduction

Pharmaceuticalresiduesintheenvironmentandtheirpossible

biologicalorsideeffectsonnon-targetorganismsarean

emerg-ingresearchinenvironmentalsciences[1].Theinterestabouttheir

occurrence,theirfateandtheirtoxicityintheenvironmentreally

tookoffattheendof1990sandthenumberofpublicationshas

beenconstantlyincreasingsincethen[2].

∗ Correspondingauthorat:UniversitédeToulouse,INP-ENSIACET,CNRS, Labora-toiredeGénieChimique,4alléeEmileMonso,F-31432ToulouseCedex4,France. Tel.:+33534323628;fax:+33534323697.

∗∗ Correspondingauthor.

E-mailaddresses:jordan.seira@ensiacet.fr(J.Seira),

caroline.sablayrolles@ensiacet.fr(C.Sablayrolles).

After administration, large fractions of pharmaceuticals are

notcompletelyassimilatedormetabolizedinthebodyandthen

excretedasparentcompoundsormetabolitesviaurineandfeces

[3]. Thesecompounds arecollectedand mixed in wastewaters,

inwhichtheirconcentrationscanreachsomemg/L[4].

Pharma-ceuticalcompoundssufferfrompartialremovalduringactivated

sludgetreatment,themostcommonwastewatertreatmentplant

(WWTP). Consequently, WWTP effluents are recognized as the

primaryspreadingsourceofpharmaceuticalpollutioninthe

envi-ronment.

Duringactivatedsludgetreatment,tracepollutantscanmainly

beaffectedbythreemechanisms:volatilization,biodegradationor

sorptiononto sludge,dependingonbothcompound andsludge

physico-chemical properties. Therefore, volatilization is usually

neglectedfor pharmaceuticals because of low Henry’sconstant

[5].Whilebiodegradationhassometimesthesignificationof

displacementofthepollutionfromtheaqueoustothesolidphase.

Monitoringtracepollutantsinsolidpartcouldbeofcrucial

impor-tancebecauseof(1)possibleinfluencetowardbioavailability(i.e.

biodegradation)tomicroorganismsand(2)stabilized-sludge

land-fillapplicationswhichcanintroducesludge-borntracepollutants

intheenvironment, increasingpotential exposurerisks.

Conse-quently,investigatingoccurrenceoftracecompoundsinbiosolids

couldbeakeyfactorfor(1)upgradingWWTPsandtrace

pollut-antsremovaland(2)theestablishmentofnewregulationswhich

areonlyfocusedonheavymetals,polychlorobiphenyls(PCBs)and

polycyclicaromatichydrocarbons(PAHs) fortracepollutants in

sludge-amendedsoilapplications.

Amongthebroadspectrumofavailablepharmaceutical

prod-ucts,there is stillone classthat paidlittleattentionin spiteof

anenvironmentallydevastating potential: theanticancerdrugs.

Includingantineoplasticandendocrine-therapydrugs,these

com-poundsaredesigned topreventor disruptcellularproliferation

incancertreatmentschemes[6].Unlikesomeothertherapeutic

classes,anticancerdrugsexhibitverydifferentphysico-chemical

properties.Tonamea few,someexamplesarelogKowranging

from–2.46to6.3andpKarangingfrom1.45to9.8[2].Mostof

theanticancerdrugspossessastrongcarcinogenic,mutagenicand

teratogenicpotentialandarethoughtasoneofthemosthazardous

contaminantsinwatercycle[7].Duetotheirmodeofaction, it

isassumed that almostalleukaryoticorganisms arevulnerable

togeneticdamagesatverylowconcentrations[8].Ashighlighted

by the literature, their consumption is increasing and trends,

includingtypeofconsumeddrugsandpracticesofconsumption,

arediversifying[6].

Themonitoring of anticancerdrugs in theenvironment has

encounteredatremendousinterestforthelast3years.

Comprehen-siveoverviewsincludinganalyticalmethodsfortheiranalysis[9],

dataabouttheirenvironmentaloccurrenceandfate[2]and

assess-mentofenvironmentalexposure[6]havebeenpublishedunder

thisperiod.Thesestates-of-artrevealedthatenvironmental

occur-renceofanticancerdrugsinwatersamplesarefewdocumentedbut

dataabouttheiroccurrenceinsolidsamplesaredefinitivelyscarce.

Althoughanalyticaldevelopmentfortheirdeterminationinliquid

samplesisstillofconcernbutfairlycommon,thereisagreatneed

ofaccurateanalyticalmethodfocusedontheirdetectioninmore

challengingmatricessuchassolidpartofsludge.

Performingextractionoftracepollutantsfromsolidmatrices

isnoteasy tohandle. Avarietyofprocedureshasbeendefined

intheliteratureandcanbedividedintwodistinctgroups:

clas-sicaland recentextractiontechniques[10].Classicaltechniques

includemechanical stirring,SoxhletandSoxtec,and ultrasound

extraction(USE),thelaterhasbeenusedonetimeforthe

extrac-tionofanticancerdrugsinsludgesamples[11].Mostofthemare

labor-intensive,time-consumingandrequirelargeamountsof

sol-vents.Theirapplicationtosolidsisnoticeablydroppedandreplaced

withmoretime-savingandeco-friendlyprocesses.Recent

extrac-tiontechniquesincludemicro-waveassistedextraction(MWE)and

pressurizedliquid(including hotwater)extraction(PLE) among

many examples. A comprehensive overview about the

extrac-tionoftracepollutantsfromsludgeaccordingdifferentextraction

techniquesisavailable[10].Duetotheincreasingnumberof

pub-lishedpapers,PLEanditsderivativesappearasthemostpromising

techniqueforefficientextraction[12,13].Uptonow,onlyone

appli-cationofPLEhasbeenreportedfortheextractionofanticancer

drugsinsludgesamples[14].

Dependingontheextractiveconditionsapplied,therecoveryof

variableamountsofco-interferingcompoundsduringPLEis

possi-ble[15].Toaddressthiswell-knowndrawback,aclean-upextract

isoftenrequired.Inmostofcases,thisstepisperformedby

solid-phaseextraction(SPE).Withtheemergenceofmixed-modeSPE

implyingpolar,non-polarandionicinteractionswiththesorbent,

selectivepurificationisallowed.Thus,mixed-modeSPEcouldbe

promisingforrecoveringanalyteswithdifferentphysico-chemical

propertiesandenhancingmethodspecificity.Mixturescontaining

anticancerdrugsareusuallyseparatedbyliquidchromatography

[2,9].Thetraceleveloccurrenceofthesedrugsinenvironmental

samplesjustifiestheuseof sophisticatedsystemssuchasmass

spectrometry (MS) detection. Thus, ultra high pressure liquid

chromatography–tandem mass spectrometry (UHPLC–MS/MS)

appearsasthemostpowerfulandadequatetoolforfastseparation

andveryselectiveandsensitivedetectionincomplexmatrices[10].

Inlightoftheseconcerns,oneofourobjectiveswastodevelop

andvalidateananalyticalmethodfordeterminingtheoccurrence

ofanticancerdrugsinthesolidpartofsludge.Thedrugsofinterest

arethealkylatingcytotoxicscyclophosphamide(CP)andifosfamide

(IFO) and theantiestrogen hormonally activetamoxifen (TAM).

Someof theirrelevantphysico-chemicalpropertiesaregivenin

Fig.1.Amongalltheanticancerdrugs,theinvestigationoftheirfate

hasrecentlybeendefinedaspreferentialduetotheirconsumption

data,theirbehaviorinWWTPandrelatedpredictedenvironmental

concentrations(PEC)intheliterature[6].Themethodisbasedon

theextractionfromthesolidmatrix usingsemi-exhaustivePLE,

extractclean-upusingtandemOasisMAX/MCXselectiveSPEand

analysisbyUHPLC–MS/MS.Tohighlighttheinfluenceof

experi-mentalconditions,optimizationofPLE parameterswasrealized

accordingtoanexperimentaldesign.Themethodwasvalidatedfor

sevensludgesamplesrepresentativeoftheFrenchWWTPsprofile.

Someeffortshave beencarriedouttoidentifywhich analytical

step wasdetrimentalin thedetermination of anticancerdrugs.

Theuseofdeuteratedstandardshasalsobeenappliedtocheckfor

possiblecompletecompensationoflossesthroughtheanalytical

procedure.Toourknowledge,thisisthefirsttimethatanticancer

drugs have beeninvestigated in sludge originated fromFrench

WWTPs.

2. Experimental

2.1. Analyticalstandardsandchemicals

Analytical standards cyclophosphamide monohydrate (CP),

ifosfamide (IFO), tamoxifen (TAM) were purchased from

Sigma–Aldrich(Saint-QuentindeFallavier,France)anddeuterated

cyclophosphamide-d4(CP-d4),ifosfamide-d4(IFO-d4),

tamoxifen-d5 (TAM-d5)werepurchased fromToronto Research Chemicals

(NorthYork,Ontario,Canada)aschemicalpowders.

Methanol(MeOH),acetonitrile(ACN)andacetonewereHPLC

grade and purchased from Scharlau (Spain). Hydrochloric acid

(HCl)37% and formic acid(HCOOH) 99% werepurchased from

VWRProlabo(Fontenay-sous-Bois,France).Ammoniumhydroxide

(NH4OH)35%waspurchasedfromFischerChemical

(Loughbor-ough,Leicestershire,UK).Ammoniumacetate(NH4CH3COO)98%

waspurchasedfromMerck(Damstadt,Germany).Na2EDTAwas

purchasedfromICNBioMedicals(Aurora,OH,USA).Theultrapure

waterusedforlaboratorypurposesaswellasLCmobilephasewas

producedfromdemineralizedwaterbyaMilliPoresystem

(Mol-sheim,France).

Stocksolution(∼1000mg/L) ofeachindividualstandard was

preparedevery4monthsbydissolvingtheappropriateamountin

MeOH.Beforeanyexperiment,workingsolutions(i.e.dilutionof

thestocksolution)werepreparedinMeOHtotherequired

con-centration.TwodistinctmixturesofstandardsCP(∼2mg/L),IFO

(∼2mg/L),TAM(∼0.5mg/L)anddeuteratedCP-d4(2mg/L),IFO-d4

(2mg/L),TAM-d5(0.3mg/L)werepreparedinthisway.For

con-venience,thetermsMIXStandardsandMIXDeuteratedwillnow

beusedthroughoutthisdocument.Tominimizedegradationof

standards,stockandworkingstandardssolutionswerewrapped

Fig.1.MRMchromatogramofspikedFS IMBRsludgesample.

2.2. Analyticalprocedure

Determiningtheanticancercompoundsinsludgewascarried

outaccordingtoaprocedureofseveraldeterminativesteps

includ-ing sample pre-treatment, extraction, purification and analysis

(Fig.2).

2.2.1. Samplecollectionandpre-treatment

Sludgesamplesusedinthisstudywereoriginatedfromdifferent

full-scaleorpilot-scaleWWTPsinMidi-Pyreneesand

Languedoc-Roussillonregions(France).Sampleswerecollectedduringgrab

samplingcampaignsbetweenMarch2009and November2011.

Foreachsamplingcampaign,asufficientamountofsludge(>5L)

was retrieved and transferred to polypropylene cans. Samples

wereoriginatedfromthreeconventional activatedsludge(CAS),

one full-scale (FS) internal membrane bioreactor (IMBR), two

pilot-scale(PS)IMBRandexternalMBR(EMBR)andonethickened

primary–secondary(TPS)sludge.DetailsaboutWWTPsandsome

relatedfeaturessuchassludgeacronymsusedthroughoutthis

doc-umentaregiveninTable1.Alltheabovementionedsampleswere

characterizedanddistinguishedaccordingtovolatilesuspended

solid (VSS)measurement. VSS wasobtainedaftercalcinationof

totalsuspendedsolid(TSS)at525◦_{Cduring2hinafurnace.TSS}

measurementwasdeterminedbyfiltrationofaknownvolumeof

sludgeaccordingtoAFNORregulationNFEN872[16].

Brieflyaftertransporttothelaboratory,eachsludgesamplewas

allowedtosettleandsupernatantwasdiscarded.Theremaining

sludge was then centrifuged to ensure a complete separation

betweenparticularandaqueousphases.Agreat amountof

set-tled sludge(1L foreach run)wascentrifugedduring 20minat

5000×gwithaMegafuge40RcentrifugefromFischerScientific

Centrifugaon

Aqueous phase discarded

0.35 g of sludge

PLE cell preparaon

15-mL extract

Dissoluon with 200 mL UHQ water pH 12

Addion of EDTA 0.1% (w/w)

Eluon with 4 mL MeOH and 6 mL acetone

Evaporaon to 2 mL

Dissoluon with 70 mL UHQ water pH 2

Eluon of neutrals with 4 mL MeOH

Eluon of basics with 6 mL NH4OH 2% in acetone

Evaporaon to dryness

Reconstuon to 1 mL and filtraon at 0.45 µm

TurboVap evaporaon to 5 mL-extract

Oasis MAX 150 mg cartridge

Oasis MCX 150 mg cartridge

UHPLC-MS/MS

Raw sludge sample

Freezing, lyophilisaon and grinding

PLE

MeOH/water (65/35), 85 bar, 100 °C, 9 min, 4 cycles

Pretrea

tmen

t

Extraco

n

Purificaon

1.

Analy

sis

Evaporaon

Purificaon

2.

Fig.2.Methodologyappliedforthedeterminationofanticancerdrugsinsludge.

werecombined and frozen at −20◦_{C. Iced sludge pellets were}

thenfreeze-driedatobscurityand−60◦_{Cunder0.045barvacuum}

(ChristAlpha1-2LD,BioblockScientific,Illkirch,France),ground

tothinparticles(<0.5mm)usingamortarandpestleandstoredat

−20◦_Cpriortouse.

2.2.2. Sampleextraction

A Dionex accelerated solvent extraction (ASE) 200 device

(Dionex,Sunnyvale,USA),whichisthetradenameforPLE,was

usedfortheextractionofanticancerdrugsfromsludge.

At the bottom of each extraction cell, one glass-fiber filter

(Dionex,Voisins-le-Bretonneux,France)wasplacedtoensurethe

filtrationofsemi-aqueousextracts.Athinsandlayer(Fisher

Sci-entific,Loughborough,UK)wasthenappliedforpre-filtration.The

driedbiosolidsamplewasweighted(0.35g),spikedwith100mL

ofMIXDeuteratedandmixedthoroughlywithsandasdispersing

agenttopreventaggregationduringextractionprocessandreduce

clumping and channeling. The ratio betweensample and sand

weightwasabout0.04.Themixturewasthenplacedinthe

extrac-tioncellandcoveredwithanadditionallayerofsand.Thecellwas

notcompletelyfilledwithsand.Adeadspaceabout0.25cmwas

lefttokeepthreadsandsealingsurfacessafe.Toallowmore

repre-sentativeadsorptionofspikedanalytesinsludge,builtcellswere

leftatroomtemperatureforaminimumof24hbeforeextraction.

Theextractionsolventandoperatingconditionswereoptimized

accordingtoamultivariateexperimentaldesignshortlydetailedin

thispaper.MeOH/ultrapurewatermixture(65/35,v/v)wasused

asextractionsolvent.Theoperatingconditionswereasfollows:

extractionpressure,85bar;extractiontemperature,100◦_C;no

pre-heatperiod;staticextractiontime,9min;numberofstaticcycles,

4;flushvolume,60%ofthecell;purgetime,120s.Thisprocedure

ledtoafinalextractvolumeof15±2mLforallthesamples.

2.2.3. Extractclean-up

Extracts were transferred to rocket-shaped bottles (200mL)

andevaporatedtoaround5mLwithaTurboVapIIconcentration

Table1

Featuresofsludgesusedinthisstudy.

Sludge Scale Personequivalent Organicload Technology pH VSS(%)

FSVLCAS Full 300000 Verylow CAS 8.3 79

FSLCAS Full 2000 Low CAS – 83

FSMCAS Full 800000 Medium CAS 7.25 91

FSIMBR Full 9000 Verylow InternalMBR 7.55 75

LSEMBR Lab(20L) – Lowa _{ExternalMBR 7.7 84}

LSIMBR Lab(15L) – Lowb _{InternalMBR 7.5 89}

TPS Full >30000 – Thickener 7.8 71

CAS:conventionalactivatedsludge;MBR:membranebioreactor;VSS:volatilesuspendedsolids;FS VLCAS:FullScaleVeryLowCAS;FSLCAS:FullScaleLowCAS;FSMCAS: FullScaleMediumCAS;FSIMBR:FullScaleInternalMBR;LSEMBR:LabScaleExternalMBR;LSIMBR:LasScaleInternalMBR;TPS:thickenedprimarysecondary.

a_{Semi-syntheticinfluentusingsamewastewaterasFSMCAS.} b _{FedwiththesamewastewaterasFSMCAS.}

workstation (Caliper Life Sciences, Hopkinton, USA) operating

at30◦_{Cundera nitrogenN}

2 pressureof1bar.Theevaporation

lasted2h.Theclean-upprocedurehasalreadybeensubmittedfor

aqueoussamples[17]andwasadaptedtoourpurposes.Clean-up

hasbeencarriedoutusingselectiveSPEtandemapproachOasis

MAX/MCX cartridges from Waters (Saint-Quentin-en-Yvelines,

France). The solvent mixtures used for SPE were prepared

everyweek.

The5-mLextractwasdissolvedin150mLofultrapurewater.

ASEvialcollectionwasalsorinsedwith50mL(5×10mL)ofultra

purewaterandtransferredtothemixtureforafinalvolumeabout

200mL.SamplepHwasadjustedto12withNH4OH35%andmixed

thoroughlywithEDTA5%(0.01%inthesample,w/w).AMAX

car-tridge(6cm3_{,150mg)wasinitiallyconditionedwith4mLofMeOH,}

4mLofacetoneand4mLof NH4OH0.5%.A 70-mLSPE

propyl-enesamplereservoirfromMacherey-Nagel(Hoerdt,France)was

stackedonthecartridgebeforeloadingthesampleataflowrateof

1mL/min.Awashsolutionof4mLNH4OH0.5%inMeOH/ultrapure

watermixture(5/95,v/v)wasappliedandfollowedbytheelutionof

targetedanalyteswith4mLofMeOHand6mLofacetonecollected

inasamefraction.Thevolumeofthefractionwasconcentrated

downto2mLanddissolvedin70mLofultrapurewatercorrected

atpH2withHCl37%.AMCXcartridge(6cm3_{,150mg)wasthen}

conditionedwith4mLofMeOH,4mLofacetoneand4mLofultra

purewateratpH2.Thesamplewasloaded(1mL/min)ona

70-mLSPEpropyleneadaptator.Thecartridgewasrinsedwith4mL

ofMeOH/ultrapurewater(pH2)mixture(5/95,v/v).Theexcess

waterpresent inthecartridgewasremovedwithastrong

vac-uumduring15minandthesorbentwascompletelydriedunder

N2streamduring20min.TheelutionofneutralsIFOandCPwas

performedwith4mLofMeOHfollowedbytheelutionofbasicTAM

with6mLofNH4OH2%inacetoneintwodistinctfractions.Details

ofSPEprocedure,retentionmechanismsandinterestofcartridges

combinationaregivenelsewhere[17].Thevolumeoftheextracts

wasreduceddownto1mLandtransferredtovialsfromAgilent

Technologies(Massy,France).Theextractswerethenevaporated

todrynessandredissolvedin1mLof(A)/(B)mobilephasemixture

(75/25,v/v)(seeTable2forcomposition)usingavortexapparatus

fromFischerScientific(Illkirch,France).AfiltrationonaSpartan

RC0.45mmsyringefilterfromVWR(Fontenay-sous-Bois,France)

wasperformedforeachextract.Theextractswerefinallystoredat

4◦_{Candobscurityduringamaximumdurationof7dayspriorto}

analysis.

2.2.4. UHPLC–MS/MSanalysis

LCseparationwascarriedoutusinganUltimate3000UHPLC

SystemfromDionex(France).Thecolumnusedforseparationwas

anACQUITYUPLC®_BEHC

18(50mm×2.1mm)witha1.7mm

par-ticlesizediameter(Waters,Saint-QuentinenYvelines,France).All

detailsaboutLCconditionssuchasinjectionvolume,flowrate,auto

samplerandcolumntemperatures,elutiongradientaregivenin

Table2.

Table2

Liquidchromatographyconditions.

Parameter Appliedcondition

Injectionvolume 10mL

Flowrate 400mL/min

Autosamplertemperature 15◦_C

Columnoventemperature 25◦_C

Mobilephase EluentA EluentB

Ultrapurewater/ACN(90/10,v/v) NH4CH3COO1mM

HCOOH0.3%

PureACN

LCgradient %EluentA %EluentB

Time(min) 0 100 0 0.5 100 0 2 78 22 3.5 77 23 4 0 100 6 0 100 8 100 0 10 100 0

Detection was achieved with an Applied Biosystems Sciex

QTRAP®_{hybridlinearion-traptriplequadrupolemass}

spectrom-eter(FosterCity,USA)equippedwithaTurbolon-SprayInterface.

TheinstrumentwasoperatedinElectroSpray(ESI)positive(+)in

MultipleReactionMonitoring(MRM)mode(dwelltime,80ms).

Theoperatingparameterswere:capillaryvoltage,5000V;source

temperature,450◦_C;gasN

2;curtaingas,20;Ionsourcegas1,20;

ionsourcegas2,70.Beforeanyexperiment,asoftcleaningofthe

coneentrancewasperformedtomaintaintopinstrumental

per-formance.Foreachcompound,conevoltageandcollisionenergies

ofthemaintransitionswereoptimized.MSandMRMconditions

are summarized in Table3.For MS spectraand chromatogram

acquisitionandexploitation,Analyst1.6.1softwarefromApplied

BiosystemsSciex(FosterCity,USA)wasused.

Aminimumof3identificationpointswereappliedto

unam-biguouslyidentifytheanalytes inenvironmentalsamples.Each

compoundwascharacterizedaccordingto(1)itsretentiontime

tRincomparisonwiththecorrespondingstandardforeachbatch

processwithatoleranceof±5%,(2)themonitoringoftwo

transi-tionsperanalyteand(3)itspresenceinoneofthe2SPEextracts.A

typicalchromatogramoftargetedanalytesinrealsampleisgiven

inFig.1.

Forquantification,MRMtransitionswereused.Six-point

cali-brationcurvesweregenerated.Fromworkingsolutions,identical

amountsofdeuteratedanalyteswereaddedtothecalibration

stan-dards,whichcontainedrelatedanalytesinconcentrationspanning

about2ordersofmagnitude.Thecalibrationstandardswere

evap-orated to dryness, redissolved in 1mL of (A)/(B) mobile phase

mixture(75/25,v/v) and filteredat 0.45mm. Calibrationcurves

Table3

MSandMRMconditionsusedtoidentifyandquantifypharmaceuticals.

Pharmaceutical Detection Transitions(m/z) DPa_{(V) EP}b_{(V) CE}c_{(V) CXP}d_(V)

IFO Positive 261.1>92.0(Q) 65 10 30 12 261.1>153.8(q) 65 10 24 12 CP Positive 261.1>139.8(Q) 65 10 27 12 261.1>105.9(q) 65 10 22 12 TAM Positive 372.4>72.0(Q) 65 10 40 15 372.4>128.9(q) 65 10 35 15

Q:quantificationtransition;q:confirmatorytransition.

a_{Declusteringpotential.} b_{Entrancepotential.} c _{Collisionenergy.} d _{Collisioncellexitpotential.}

wereperformedatthebeginningofeach batchprocess.Curves

werebuiltbycalculatingtheratiosbetweenthepeakareaofeach

analyteandthepeakareaofcorrespondingdeuteratedstandard

usingweighted1/xmodelforlinearregression.Alongthesequence,

qualitycontrol(QC)sampleswerealsoanalyzedtoconfirmtheir

validity.QCsampleswereahigh-andlow-concentrationlevelof

thecurves(1orderofmagnitude).Nosignificant(<12%)deviation

hasbeenobserved.Assludge extractsmaycontentmany

inter-feringcompounds,blanksamples(mobilephasemixturewithout

analytes)wereincludedevery5injections.Nocross-contamination

hasbeenobserved.Attheendofeachsequence,chromatographic

columnwaswashedthoroughlywithacidifiedwater(pH3)and

pureACN.

Instrumentaldetectionlimits(IDL)andinstrumental

quantifica-tionlimits(IQL)weredeterminedbyserialdilutionofeachstandard

downto2pginjected.TheIDLandIQLweresetasasignal-to-noise

(S/N)ratioof3and10ofthechromatographicresponse

respec-tively.

2.3. Methodperformances

Theperformancesoftheanalyticalprocedurewereevaluatedfor

eachanalytethroughtheestimationofmethodefficiency,

repeat-abilityandreproducibility,sensitivityandmatrixeffect.Estimation

ofthelinearitywasalsoconsideredaspartofthevalidation.

2.3.1. Validationprocedure

Todemonstratetherobustnessoftheanalyticalprocedure,the

sevensludgesamplesdefinedinTable1weresubmittedtothe

validationprocess.Foreachfreeze-driedbiosolid,4sampleswere

spikedwith100mLofbothMIXStandardsandDeuteratedand1

samplewasspikedwith100mLofMIXDeuteratedfornative

ana-lyteconcentration.Allthe sampleswerethensubmittedtothe

previouslydescribedprotocol.Thisexperimentalset-upallowsfor

thedeterminationof theefficiencyof theentireprocedure(i.e.

methodefficiencyMEff)andnotforeachanalyticalstep.The

deter-minationoftheMEffwascalculatedfollowingEq.(1):

methodefficiencyMEff (%)=Qpreextract−Qback

Qspike

×100 (1)

whereQpreextractistheamountintheextractaftercomplete

pro-cedure (ng), Qback is the amountpresent in the native sample

(backgroundquantity)(ng)andQspikeisthequantityofthespike

(ng).

For three freeze-dried sludges (FSLCAS, FSMCAS, FSIMBR),

MEffwasalsodeterminedover arangeof4concentrations.For

eachsludgecandidate,4sampleswerespikedwith100mLofMIX

DeuteratedanddifferentvolumesofMIXStandards(10,50,100,

200mL)toachieveconcentrationsinthesamplesof60,300,600,

1200mg/kgofdrymatter(DM)andthensubmittedtotheentire

protocol.Measuredanalyteconcentrationswereplottedasa

func-tionoftheirrelatedspikedconcentrationsandthecorresponding

slopewasdetermined(Slopeplotted).Four-concentrationMEffwas

determinedforeachanalyteaccordingtoEq.(2):

four-concentrationmethodefficiencyMEff (%) = Slopeplotted

Slopecalibration

×100 (2)

whereSlopeplottedistheslopepreviouslydefined,Slopecalibration is

theslopeofthecalibrationcurve.Inbothexperiments,absolute

andrelativeMEffwerecalculated.ForrelativeMEff,allthevalues

werecorrectedrelativetothedeuteratedanalogues.

Repeatability(intra-dayprecision)wasexpressedastherelative

standarddeviation(RSD,%)obtainedfromtheMEffexperimentat

asingleconcentrationandextracted,purifiedandanalyzedinthe

samebatch.Reproducibility(inter-dayprecision)wasdefinedand

conductedinthesameconditionsbutondifferentbatchesandwas

determinedonlyforthreefreeze-driedsludges(FSLCAS,FSMCAS,

FSIMBR).

Thesensitivityoftheanalyticalmethodwasdetermined

accord-ingtothedefinitionsofmethoddetectionlimits(MDL)andmethod

quantificationlimits(MQL).MDLandMQLwerecalculatedusing

Eq.(3):

methodlimitsML (mg/kgDM)=

IL×Vextract

MEffabs×m

(3)

whereIListheconsideredinstrumentallimit(mg/L),Vextractisthe

volumeofthefinalextract(=1mL),MEffabsistheabsolutemethod

efficiencycalculatedforasingleconcentration(0<MEffabs<1),mis

thedriedsampleweight(=0.35g).

2.3.2. Analyticallimitation

Toevaluatetheperformancesofeach analyticalstep,

freeze-driedsamplesandsubsequentextractswerespikedatdifferent

stepsofthe procedurewith100mLof bothMIXStandards and

Deuterated.Theexperimentalschemewasinspiredfromthe

lit-erature[18]andconductedintriplicateforFS MCASandFS IMBR.

Spikeswereapplied:

(a)Beforefreeze-drying onrehydrated freeze-dried samplesto

assesstrueMEff;

(b) Beforeextractiontoevaluatethecombinedrecoveryof

extrac-tion,purificationandanalysis(MEffdefinedinSection2.3.1);

(c) BeforepurificationonOasisMAXtoevaluatetherecoveryof

bothpurificationandanalysis;

(d)BeforepurificationonOasisMCXtoevaluatetherecoveryof

secondpurificationandanalysis;

(e)Beforeanalysistoevaluatetherecoveryoftheanalysis.

Absoluteandrelativerecoveriesweredeterminedinthesame

wayas forMEffestimation. Thefollowing Eq. (4) wasusedfor

calculation:

recovery (%)=Qstep−Qback

Qspike

×100 (4)

whereQstepistheamountinthefinalextractafterspiketothe

cor-respondinganalyticalstep(ng).Forrelativerecovery,allthevalues

werecorrectedrelativetothedeuteratedanalogues.

Thespikingprocedureappliedin(e)alsoallowsforthe

deter-minationofmatrixeffect(ME),accordingtoEq.(5):

matrixeffectME (%)=





whereApostextractistheareaintheextractspikedjustbeforethe

analysis,Abackistheareaintheextractofnativeunspiked

sam-ple(backgroundarea)andA_spikeistheareaofthecorresponding

spike.AbsoluteMEcalculationwasbasedontheareaofanalyte

withoutcorrectionwhilerelativeMEwascalculatedrelatedtothe

deuteratedanaloguearea.

Theaccuratedeterminationoftherecoveriesforeach

analyt-icalstep waspossible.Theefficiency ofeach detailed step was

determinedaccordingtoEq.(6):

analyticalstep n efficiency (%)= Rn

Rn+1

×100 (6)

whereRistheabsoluteorrelativerecovery(%)atagiven

spik-ingstep,nisavaluerangingfrom1to4anddescribingaspecific

(n=1)pretreatment(i.e.freeze-drying)bycomparingexperiments

(a)and(b)

(n=2)extractionbycomparingexperiments(b)and(c)

(n=3)purificationI(i.e.OasisMAX)bycomparingexperiments(c)

and(d)

(n=4)purificationII(i.e.OasisMCX)bycomparingexperiments

(d)and(e)

3. Resultsanddiscussion

3.1. OptimizationofPLE

3.1.1. Selectionofextractionsolvent

Thesolventmustbeabletosolubilizethetargetedanalytesfrom

thematrixwithfewinterferingcompoundsasfaraspossible.Since

theanalytesvaryinphysico-chemicalproperties,thechoiceof

sol-ventmixtureswascrucialbutalsolimited.Ourstrategyforselecting

mixturesrelieson(1)solventspreviouslyappliedwithsuccessin

theliteratureand(2)closepolaritymatchingbetweenanalytesand

solventmixtures.Differentpureandbinarysolventsweretested.

Puresolventswereacetone,MeOH,ACN,water(pH7)andbinary

mixtureswereacetone/ACN(1:1),MeOH/ACN(1:1),acetone/water

(1:1),MeOH/water(1:1)and ACN/water(1:1).Asnodetectable

concentrationoftargetedanticancerdrugswasmeasured,FS LCAS

sludgewasselected,spikedwith100mLof bothMIXsand

sub-mittedtothewholeanalyticalprocess.Alltheexperimentswere

performedinduplicate.InitialPLEconditionswereappliedfrom

theliterature:extractionpressure,138bar;extraction

tempera-ture,100◦_{C;nopre-heatperiod;staticcycleextractiontime,5min;}

numberofstaticcycles,3;flushvolume,60%ofthecell;purgetime,

120s[14].Thesolventmixtureefficiencywasinvestigatedby

com-paringthemeanareasoftargetedanalytesforeachtestedcondition

(datanotshown).Areasofdeuteratedanalogueswerealso

com-pared.Inthesametime,extractioncellsfilledwithdispersingagent

werespikedandextractedinthesameconditionstoinvestigate

thethermaldegradationofanalytes.Nosignificantlossesoccurred

underchosenparameters,thusconfirmingthestability.

Forthetestedsolvents,alltargetedanalyteswererecoveredin

differentamounts.Extractsexhibitingdifferentaspectswerealso

obtained.Pureandmixedorganicsolventsledtohighlycolored

andclearextractswhilewaterledtobrownandveryturbidones.

Semi-organicmixturesgaveintermediateprofiles.Turbidaqueous

sampleswereresponsibleforthecloggingofthecartridgeduring

thepurification.Consequently,water(pH7)wasnotselectedas

extractionsolventinourexperimentalscheme.Higherareaswere

obtainedforIFOandCPusingMeOH/water(1:1)andforTAMusing

pureMeOH.Nodiscrepancieswereobservedfordeuterated

ana-loguesareas.ACNandderivedmixturesgavetheworstresultsfor

eachcompound.ThelowerefficiencyofACNforextracting

pharma-ceuticalsfromsolidsampleshasalreadybeenreported[13,18,19].

Unsurprisingly,watermixtureswereefficienttoextractpolar

ana-lytes IFO and CP while pureorganic solvents were efficientto

extractapolarTAM.AsTAManalysiswasmoresensitivethanfor

IFOandCP,MeOH/waterasextractionsolventwasfoundtobea

goodcompromise.Fromtheliteratureandourfindings,the

supe-riorcapabilityofMeOH/watermixturetoextractpharmaceuticals

fromsolidsampleshasbeenfound[13,18–23].

3.1.2. Optimizationusingexperimentaldesign

ThenumberofparametersaffectingPLEisveryhighsotheone

variableatatime(OVAT)strategywasnottoconsiderhere.Finding

thebestoperatingconditionsformaximizingrecoverieswithfew

experimentswasachievedusingacentralcompositedesign(CCD).

Accordingtotheliterature,theparametersofinterestwerethe

sol-vent(MeOH/water)ratio(variableA),theextractiontemperature

(variableB),theextractionpressure(variableC),thestaticcycle

duration(variableD)andthenumberofcycles(variableE)[24].

TheCCDconsistedinafractionalfactorialdesignincludingthefive

variablesattwolevels(25−1_{),eachaugmentedbytenstarpoints}

and6centerpoints.Thetotalnumberofextractionswas32.The

lowandhighlevels(domainboundaries)foreachparameterwere

commonPLEvaluesdeterminedfromtheliterature[10,24].These

valueswere10–90%(MeOH/waterratio),70–110◦_{C(temperature),}

70–130bar(pressure),4–16min(cycleduration)and1–5

(num-berofcycles).Thecompletedefinitionoftheexperimentaldesign

appliedisgiveninSupplementaryContent1.FSLCASsludgewas

chosenforoptimizationasnotargetedanalyteshavebeendetected.

Toevaluatetheefficiencyofextraction,100mLofMIXStandards

werespikedpriortoextractionand100mLofMIXDeuteratedwere

spikedintothecorrespondingextract.

Supplementary material related to this article found, in the

onlineversion,athttp://dx.doi.org/10.1016/j.chroma.2013.01.114.

Therecoveriesobtainedforeachanalyteandexperimentare

giveninSupplementaryContent2.Someyieldsweresuperiorto

100%whichcouldbeattributedtomethoderrors,sludgesample

inhomogeneity[25]orsignalionenhancementduringanalysis.In

thedefinedexperimentaldomain,TAMdisplayedstrongvariability

withvaluesrangingfrom0to205%.Moreover,thevariabilitywas

remarkablyhighforthe6center points(experiments27–32).It

suggestedthatTAMextractionwasaffectedbyanunconsidered

parameteroranyotherunknownprocess.Asimple experiment

wasconductedbywashing thoroughlywithorganicsolventthe

laboratoryvesselandanalyzingthesolvent.Quantifiableamounts

ofTAMhavebeenmeasured,confirmingadsorptionphenomena.

DeterminationofTAMwasthereforenotpossible.Fortheother

analytes,thevariabilityatthe6centerpointshasbeendetermined

(SupplementaryContent2).IFOexhibitedlessvariabilitythanCP

witharelativestandarddeviation(RSD)of6%versus13%.

Supplementary material related to this article found, in the

onlineversion,athttp://dx.doi.org/10.1016/j.chroma.2013.01.114.

TheMinitab®_{softwarewasusedforthestatisticalstudy.Owing}

totheCCD,thecoefficientsofasecondorderpolynomialmodel

describingtheeffectsofthe5variablesonIFOandCPrecoveryhave

beenestimated.Thetwomodelsadequatelyrepresentedthedata

aslack-of-fitp-valuesweresuperiorto0.05(0.21forIFOand0.26

forCP).Thecorrelationbetweenpredictedandobservedrecoveries

wasupto99%forIFOand98%forCP.

Inordertoseewhichvariables(i.e.parameters)werethemost

influent ontheresponse, standardizedPareto chartswere

con-structedandaregiveninFig.3.TrendsbetweenIFOandCPwere

rathersimilar.Inbothcases,thesolventratiowasprobablythemost

determiningfactorforextractionefficiency.However,itsinfluence

wasdifficulttoassess,asthis parameterwasimpliedinseveral

significantinteractionssometimesofopposite trends.Indeed,it

appearedthatsomeinteractionsbetweenparameters,suchasA*D

andA*Eforexample,werestronglyinfluent.Itmeansthat

varia-tionsinextractionrecoverywerenotstrictlyassignedtoasingle

parameterbutcouldalsobeduetosynergisticeffectsoftwoor

morevariables.Theseresultsjustifytheuseofexperimentaldesign

ratherthanOVATstrategy.

Ourobjectivewastodeterminethebestvaluesofthefive

param-etersthatallowarecoveryofaround100%witha5%tolerance.Due

tothesecondorderofthemodels,aninfinitecombinationofthe

factorsallowstoreachthisgoal.Soresponsesurfacemethodology

wasusedtodeterminetheareawherethecriterionisfulfilled.The

valuesofthefiveparameterswerechosenintheseareas,taking

intoaccountthefollowingexperimentalconsiderations.

First, aqueous or highly aqueous extracts were not

recom-mended in our experimentalscheme due to possible cartridge

clogging.Moreover,themorepolarthesolventmixture,theless

Fig.3. StandardizedParetocharthighlightingtheeffectofPLEparametersinappliedexperimentaldesignforIFO(up)andCP(down).Theverticalstraightlineisthelimit ofsignificance.

waspreferential(middleofdomain).Then,theapplicationofhigh

temperaturein PLE decreasestheviscosity ofthe solvent,thus

allowingitsbetterpenetrationintosamplematrixandincreasing

itscapacitytosolubilizetheanalytes[20].Fasterextractionrates

arealsoexpectedwithhightemperatures[15].Nevertheless,high

temperaturecouldalsoleadtolossinmethodselectivityduetothe

extractionofmoreco-extractablecompounds[20].Relativelyhigh

temperaturewasthuspreferential(upperpartof domain).Next,

pressureseemedtobethelesssignificantparameter,whichisa

commonfindingintheliteratureforPLE[13,25,26].Itsroleisto

maintainthesolventintheliquidstateatextractiontemperature.

Lowpressurewassufficient(lowerpartofdomain).Finally,the

dura-tionandnumberofcyclesweredeterminedsimultaneously.Long

cycletimecouldleadtoabetterdiffusionofanalytesbutthe

multi-plicationofshortcyclecouldbefavorabletorecovery[20].Indeed,

theintroductionof freshsolventat eachcyclecouldallownew

equilibriumbetweenanalytesandsolvent,whichcouldbe

inter-estingfor stronglyentrapped analytes.Consequently,low cycle

duration(lowerpartofthedomain)and manycycles(upperpart

surfaceresponseswereplottedand displayedinSupplementary

Contents3and4.Thechosenexperimentalconditionswerethe

following:MeOH/water65/35(v/v),extractiontemperature100◦_C,

extractionpressure85bar,staticcycleduration9minand4cycles.

Supplementarymaterial related tothis article found, in the

onlineversion,athttp://dx.doi.org/10.1016/j.chroma.2013.01.114.

3.2. Extractclean-up

Extract clean-up was required to concentrate the analytes

and to remove the interfering components. As sludge was

expected to contain much more interferents than wastewater

samples, high sorbent weights (150, 500 and 1000mg) were

applied.Briefly, threetypesofsorbents wereselected: reversed

phase, hydrophilic–lipophilic balance (HLB) and mixed-mode

anionic-cationicexchange.FSLCASsludgePLEextractswere

gen-eratedand spikedprior topurification.Reversed phase sorbent

yielded very low recoveries for IFO and CP and were rejected.

HLB yielded better recoveries but the major part of

interfer-ing compounds were concentrated in the final extract, which

couldintroduceanalyticaltroubles(i.e.strongmatrixeffect)with

more complex sludge samples. Therefore, HLB sorbents were

rejected. In our previousstudy [17], mixed-mode anionic- and

cationic-exchangeSPEhasprovenvalueintheselectiverecoverof

targetedanalytesinsludgeaqueoussampleswithrelativelylow

matrixeffect. Thisprocedurehasbeenretained. Asthesorbent

weightforpurificationwastwotimesandahalfhigher,the

con-ditioning,washingandelutingvolumesweremultipliedbytwo.

Lightlycoloredandclearextractswereobtainedformostofthe

samples.Purificationprocedurewasthenconsideredsatisfactory.

3.3. Performancesoftheanalyticalmethod

AsnoCRMwasavailableforvalidation,in-housematerialwas

used. In-housematerial was freeze-dried sludge spikedwith a

knownamountoftargetedanalytes.Sevendifferenttypesofsludge

werestudiedtodemonstratethecompletesuitabilityofthe

proce-dure.

The linearityof the internal calibrationcurves was

satisfac-tory(R2_{>0.995) for IFO and CPover the tested concentrations}

(1–500mg/L)andvalidationperiod(2months).Indirectly,method

linearitywasalsostudiedduringMEffestimationoverfour

con-centrations(seeSection2.3.1)forFSLCAS,FSMCASandFSIMBR.

Linearitywasobserved (R2_{≥0.990)foreach analyteand sludge}

tested(datanotshown).Thus,themethodshowedgoodspecificity

fortheanalysisoftargetedanalytes.

Recoveriesofselecteddrugsfordifferenttypesofsludgeare

given in Table4. Bothabsoluteand relative methodrecoveries

weredistinguishedasrecommendedintheliterature[12].Absolute

MEffvalueswereverydifferentanddependentonthecompound

andsludgeconsidered.AbsoluteMEffrangeswere1.5–33%forIFO

and2.2–47%forCP.ForFSLCAS,FSMCASandFSIMBR,the

agree-mentbetweenMEffatasingleandfourconcentrationsvalidatethe

“single-point”procedureforeachsludge.Theabsoluterecoveries

forIFOandCPwerelimitedforallthesamples(<50%)butnot

crit-icalfortheirdeterminationduetothehighsensitivityofMS/MS

detection.Nosignificantcorrelationhasbeenfoundbetweenthe

recoveriesandsludgefeaturesaccordingtopH,VSSandthe

biolog-icalprocess(seeTable1).TheverylowmethodefficiencyforTPS

sludgeimpedesthequantitativedeterminationofIFOandCP.Since

VSSwasthelowest,othercharacteristicmightbemorerelevant

toexplaintheverylowmethodefficiency.AsTPSsludgeappeared

partiallydigested,harshchemicalsurroundingsofTPSsludgecould

havebeendetrimentalforIFOandCPrecoveryduringthe

extrac-tionorpurification.Strongmatrixeffectoccurringduringanalysis

wasalsopossible. Table

4 Analytical method performances and validation data. Sludge IFO CP MEff ± SD a(%) MEff b(%) MDL (m g/kg DM ) MQL (m g/kg DM ) MEff ± SD a(%) MEff b(%) MDL (m g/kg DM ) MQL (m g/kg DM ) Absolute Relative Absolute Relative Absolute Relative Absolute Relative FS VLCAS 29 ± 0 102 ± 3 3.9 9.9 38 ± 5 99 ± 11 3.0 7.5 FS LCAS 16 ± 1 99 ± 7 15 98 7.2 17.9 27 ± 1 100 ± 3 26 97 4.2 10.4 FS MCAS 14 ± 2 110 ± 13 15 108 7.9 19.8 24 ± 3 105 ± 12 22 100 4.7 11.8 FS IMBR 29 ± 2 104 ± 10 28 95 4 10 40 ± 1 97 ± 6 38 94 2.8 7.1 PS EMBR 33 ± 3 106 ± 4 3.5 8.8 47 ± 2 96 ± 4 2.5 6.1 PS IMBR 20 ± 1 104 ± 3 5.9 14.7 35 ± 1 98 ± 1 3.2 8.1 TPS 1.5 ± 0.2 106 ± 4 74 186 2.2 ± 0.3 92 ± 5 51 128 MEff: method efficiency; SD: standard deviation; MDL: method detection limit; MQL: method quantification limit; DM: dry matter. a Calculated for a single concentration (n = 4). b Calculated over a four-concentration range.

Forthedifferentsludges,relativeMEffvalueswereconsidered

excellent and rangedfrom 99 to 110% for IFO and from 92 to

105%forCP(seeTable4).Therefore,deuteratedstandardswere

completelysuitable for IFOand CPdetermination in each case.

Moreover,theuseofonlyonesurrogatestandardalongtheentire

protocolprovidedmoreaccurateresultsincomparisonwith

ana-lyticalmethods usingat leasttwo surrogate standards,one for

extractionandoneforanalysis,asencounteredin theliterature

[20,21].

The repeatability of the method was calculated from the

standarddeviationsgiveninTable4foreachsludge.ForIFO,RSDfor

absoluteandrelativeMEffwereintherange0.8–15%and2.4–12%

respectively.ForCP,RSDwereintherange2.4–14%and1.0–11%

respectively.These values havethesignificance of good overall

repeatability(<15%)ineachcase.Thereproducibilityofthemethod

hasbeencalculatedasthesamemannerandwasbelow14%and

consideredsatisfactory(<15%)forFS LCAS,FSMCASandFSIMBR

(datanotshown).Therefore,therobustnessoftheprocedurehas

beenproven.

ForIFOandCP,MDLrangedfrom3.9to74mg/kgDMandfrom

2.5to51mg/kgDM respectively (Table4).Withtheexception of

TPS sludge, alltheMDLs were lower than10mg/kgDM

display-inggoodoverallmethodsensitivity.Theconclusionsarethesame

forMQLslowerthan20mg/kgDM whicharethebest

quantifica-tionlimitsreportedintheliteratureforbothcompound[11].The

uncommonlylowsampleandpurificationsorbentweightsapplied

in the experimental schemewere not limiting in the

achieve-ment of low method limits, reaching possible environmental

requirements.

Intheoverall,ouranalyticalstrategyprovedgoodsensitivity,

selectivityand specificityduetothevalidationonsevensludge

samplesfromdifferentorigins.However,itisimportanttonote

thatrecoveriesobtainedforspikedsamplescouldoverestimatethe

efficiencyofthemethodforincurrednativeanalyte[25].Becauseof

limitationsindiffusionandkineticsofthesorptionprocess,spiked

analyteswillalwaysbeless retainedthanthenativeones[27],

Toassess therepresentativeness of freshlyspiked comparedto

incurredanalytes,anadditionalexperimentonPSEMBRsludgehas

beencarriedout.PSEMBRhasbeencontinuouslycontaminated

withanticancerdrugsduring80days.Thisprocedureallows

ana-lytestopenetratemuchmoreintothevolumeofthematrixrather

thanonthesurface.Sludgewassampledondays10,30and60

duringcampaign,whichcorrespondsrespectivelyto0.5,1.5and

3timesthesludge age.Eachsamplewasfreeze-dried andsplit

equallyintwo.Thesecondaliquotreceivedanadditionalspiking

of10mLofMIXStandards.Allthesampleswerethensubmittedto

thewholeanalyticalprocedure.Themeasuredconcentrationofthe

freshlyspikedsamplewascorrectedbysubtractingtheamountof

thespiketoassessthenativeconcentration.Thecorrectedvalue

wascomparedtotheconcentrationmeasuredinthesample

with-outadditionalspike.Nosignificantdifferencesweremeasuredfor

IFO(RSD<4%)andCP(RSD<3%).Itappearsthattheproposed

ana-lyticalmethodisnotspecifictofreshlyspikedsamplesandcanbe

appliedtoagedsamples.Thiscouldbeattributedtothe

numer-ousextractioncyclesinPLE,allowingtheexhaustionofthematrix

fromeasily accessiblecompartments (spiked)toless accessible

ones(incurred).ThesorptiveinteractionsofIFOandCPinfreshly

spikedandagedsamplescouldalsobecomparable.

Fig.4.(a)Meanrecoveries±standarddeviationforIFOinFSMCASsludge(up)andFSIMBRsludge(down)forthedifferentstepsoftheanalyticalprocedure(n=3).(b) Meanrecoveries±standarddeviationforCPinFSMCASsludge(up)andFSIMBRsludge(down)forthedifferentstepsoftheanalyticalprocedure(n=3).Therecoveries weredeterminedaccordingtoEq.(4).

3.4. Whichanalyticalstepisthemostlimiting?

Assludgematrixcomponentscanstronglyinfluencethe

effi-ciencyofthesampletreatmentstage,theobjectiveherewasto

determinewhetherthelimitedabsoluterecoverieswerelinkedto

asameanalyticalstageoriftheywererelatedtodifferentstages

dependingonthesludgenature.Todoso,twotypesofsludgewith

differentorganiccontent(i.e.VSS)havebeenselectedandspikedat

differentanalyticalstepsdescribedinSection2.3.2.FSMCASwas

selectedforitshighorganiccontent(91%)andFSIMBRforits

rela-tivelyloworganicone(75%).TPSsludge(71%)wasrejecteddueto

theanalyticalchallengepreviouslydescribed.

TheprofilesobtainedforIFOandCParedisplayedinFig.4aand

brespectively.Theabsoluterecoveriesdisplayedthetrueefficiency

ofthespikingstages.Evenifrecoveriesrelatedtotheanalysisare

comparableorsomewhathigherthanthoserelatedtothewhole

method,thequantificationofIFOandCPisdeeplydisturbedbythe

matrixeffect(ME)ineachsample,possiblyduetotheuseof

semi-organicsolventduringPLE.ForIFO,recoveriesassociatedwiththe

couple“Wholemethod;Analysis”are14%;45%forFS MCASand

26%;25%forFSIMBR.ForCP,recoveriesare22%;51%and38%;51%.

Theuseof(semi-)organicsolventduringPLEcouldberesponsible

fortheextractionofmanyinterferingcompoundsassuggestedin

theliterature[10,15,24]thusdecreasingclean-upefficiencyand

resultinginrelativelyhighME.

Theefficiencyof each analytical stepfromthe pretreatment

untiltheanalysishasbeencalculatedfollowingEq.(6) givenin

Section2.3.2.The resultsare displayedin Fig.5.Only absolute

recoverieswereusedforcalculation.

Fig.5. RecoveryprofilesforIFO(up)andCP(down)intwotypesofsludge.The recoveriesweredeterminedaccordingtoEq.(6).

Theprofilesareverydifferentbetweensludgesbutnotbetween

analytes.Foragivensludgesample,itsuggeststhatIFOandCPare

submittedtothesameorcloseprocessesduringeachstage.The

highvariabilityobservedforsomeanalyticalstepsisfullyexplained

bytheadditionofvariancesimpliedbyEq.(6)butnotcriticalfor

trendexplanation.

PretreatmentstagedidnotimplyanysignificantlossesforIFO

andCPineachcase.Freeze-dryingisoftenrequiredbecausewet

samples can prevent from efficient PLE [15]. Grinding ensures

shorterdiffusionpath-lengthsduringextractionandenhances

sol-vent penetration [15].Both stepscan be responsiblefor losses

butareusuallyneglectedduringmethoddevelopment.Fromour

resultitisdemonstratedthatnon-volatileanalytes,whichisthe

case of pharmaceuticals, are not sensitiveto freeze-drying and

grinding.Therefore,theuseofspikedfreeze-driedsamplesduring

methodvalidationwaseffectivelysufficient.Theextractivestepled

tosatisfactoryrecoveriesbetween78and105%ineachcase.For

sludgesamples,theversatilityoftheoptimizedPLEmethodhas

beendemonstrated.Thepurificationstageefficiencywasstrongly

dependentonthesludgenature.Forbothanalytes,higherlosses

were observed for FSMCAS sludge. It could be explained by

thenatureof interferingcompoundspresentinthePLEextract,

which may have competed for binding sites and lowering the

clean-upefficiency.Itisalsoimportanttonotethatevaporative

stepsalongtheprocedurewerenotresponsibleforanyanalyte

loss.

Intheoverall,theanalysiswasthemostlimitingfactorinthe

quantification.CPsufferedfromMEupto49%for bothsludges

while IFO suffered from ME of 55 and 75% for FSMCAS and

FSIMBRsludgesrespectively.Additionally,itappearedthatsludge

organicityaccording toVSS measurement wasnot sufficient to

explainMEasnocorrelationbetweenVSS,analytesandMEwas

found.EvenifVSSisaneasy-to-handleandquickmeasurement,it

seemsthatthecharacterizationofthesludgematterandrelated

extractcouldbemorerelevantintheunderstandingofME

ori-gins.

3.5. Applicationtoenvironmentalsamples

Optimized method was applied to the biosolid samples

describedinTable1.Measuredmeanconcentrationsaregivenin

Table5.

Except for FSLCAS, one or two of the targeted drugs were

detectedorquantifiedinoursamplesthusconfirmingthe

occur-renceofanticancerdrugsinsolidpartofsludge.Concentrationsin

solidphaseforIFOrangedfrom11.4to42.5mg/kgDMwhileCPwas

quantifiedonly inFSMCAS ata concentrationof 12.6mg/kgDM.

Thisconcentrationis ofthesame orderof magnitudethanone

reportedintheliteraturefor excesssludge[14].Fromourdata,

contaminatedsludgesaremostlythoseofWWTPstreatingeach

daylargeamounts of wastewater.It could bethoughtthat the

Table5

Anticancerdrugsconcentrationsincollectedbiosolidsamples.

Sludge Pharmaceuticalsa_(mg/kg DM) IFO CP FSVLCAS 11.4±2.1 <MQL FSLCAS <MDL <MDL FSMCAS 41±23 12.6±4.9 FSIMBR 42.5±14.6 <MQL PSEMBR <MQL <MDL PSIMBR <MQL <MQL TPS <MQL <MQL

MDL:methoddetectionlimit;MQL:methodquantificationlimit.

contaminationismuchmorerelatedtothetreatedperson

equiv-alent number than the sludge physico-chemical nature. The

quantificationofIFOinFSIMBRcouldbeattributedtoapossible

accumulationassludgeageislong(100days)andbiodegradation

isnot expected[28–32].In theoverall, verylow levelsof

anti-cancerdrugs were determined in our solid samples originated

fromdifferentWWTPs.Thisisingoodagreementwithlevelsof

concentration found or predicted in the literature [6,11,30]. It

couldbe explained bythe relatively low consumption and the

possiblelowsorptionaffinity forsludge duetohighpolarityof

IFOandCP.However,lowconcentrationsinsludgemaynothave

the significance of low toxicity for microorganisms and more.

Someotherfieldresultsarerequestedtoconfirmornotthesefirst

conclusions.

4. Conclusion

Inthispaper,anoriginalanalyticalmethodwasproposedto

recoveranticancerdrugs from solidpart ofsludge. The

experi-mentalset-upconsistsofextractionfromthesolidmatrixusing

PLE, clean-up by selective SPE and analysis by UHPLC–MS/MS.

Someeffortsfocusedontheextractionefficiency,themethod

vali-dationandtheanalyticallimitation.Theuseofanexperimental

designtooptimizetheextractionrevealedtheconcomitanteffect

ofsome parameters duringextraction, which helpedto

under-standthetruefunctioningofPLE.Thevalidationof themethod

wasappliedtosevendifferentsludgesamples.Methodvalidation

requirementsimplyinglinearity,repeatability,and

reproducibil-itywerefulfilled.Theanalyticalperformanceswereverydifferent

betweensludgesampleswithmethodefficienciesandMDLs

span-ningmorethanoneorderofmagnitude.Thus,methodvalidation

shouldbesystematicallyappliedforeachnewsampleandcouldbe

ofgreatinterestformonitoringprograms.Matrixeffectoccurring

duringanalysiswasdemonstratedasthemostlimitingfactorfor

thequantificationofeachanalyte.However,theuseofdeuterated

standardsspikedattheverybeginningwasefficienttoovercome

analytical troubles regardless of the matrix composition.

Vari-oussludgesampleswereanalyzed,confirmingtheenvironmental

occurrence of anticancer drugs in sludge. Up tonow, the

pro-posedmethodisonlythethirdanalyticalprocedureavailablein

theliteraturefor the extractionof anticancerdrugs from

envi-ronmentalsolidsamples,eachofthemdealingwithsludges.The

developedmethodisalsothemostsensitive(uptolowmg/kgDM),

detailedandversatile.Theneedofanalyticalmethodsand

environ-mentaldataaboutanticancerdrugsisstillofconcerntoestablish

theiroccurrenceinthewatercycleatnationalandinternational

scales.

Acknowledgment

ThisworkformpartoftheprojectANR-09-JCJC-0005

“BioMed-Boue”supportedbyANR(FrenchResearchAgency).

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