HAL Id: hal-00926528
https://hal.archives-ouvertes.fr/hal-00926528
Submitted on 9 Jan 2014
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of
sci-entific research documents, whether they are
pub-lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
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
Jordan Seira, Catherine Claparols, Claire Joannis-Cassan, Claire Albasi,
Mireille Montréjaud-Vignoles, Caroline Sablayrolles
To cite this version:
Jordan Seira, Catherine Claparols, Claire Joannis-Cassan, Claire Albasi, Mireille
Montréjaud-Vignoles, et al.. 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. Journal of Chromatography A, Elsevier, 2013, vol. 1283,
pp. 27-38. �10.1016/j.chroma.2013.01.114�. �hal-00926528�
To link to this article
: DOI:10.1016/j.chroma.2013.01.114
http://dx.doi.org/10.1016/j.chroma.2013.01.114
This is an author-deposited version published in:
http://oatao.univ-toulouse.fr/
Eprints ID:
9948
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
O
pen
A
rchive
T
oulouse
A
rchive
O
uverte (
OATAO
)
OATAO is an open access repository that collects the work of Toulouse researchers and
makes it freely available over the web where possible.
Any correspondence concerning this service should be sent to the repository
administrator:
staff-oatao@listes-diff.inp-toulouse.fr
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
a,b,∗,
Catherine
Claparols
c,d,
Claire
Joannis-Cassan
a,
Claire
Albasi
a,
Mireille
Montréjaud-Vignoles
b,
Caroline
Sablayrolles
b,∗∗aUniversitédeToulouse,INP-ENSIACET,CNRS,LaboratoiredeGénieChimique,4alléeEmileMonso,F-31432ToulouseCedex4,France bUniversitédeToulouse,INP-ENSIACET,INRA,LaboratoiredeChimieAgro-Industrielle,4alléeEmileMonso,F-31432ToulouseCedex4,France cCNRS,LaboratoiredeChimiedeCoordination,205routedeNarbonne,BP44099,F-31077ToulouseCedex4,France
dUniversitédeToulouse,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 com-plete elimination, sorption onto sludge can beconsidered as a
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 inaluminumandstoredat−20◦Cafterpreparation.
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 (Illkirch,France) operatedatsludgetemperature.Allthepellets
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.
aSemi-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) EPb(V) CEc(V) CXPd(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. aDeclusteringpotential.
bEntrancepotential. 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 (%)=
Apostextract−Aback Aspike −1 ×100 (5)whereApostextractistheareaintheextractspikedjustbeforethe
analysis,Abackistheareaintheextractofnativeunspiked
sam-ple(backgroundarea)andAspikeistheareaofthecorresponding 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 analyticalstep.Thus,thecorrespondingstepsare:
(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 selective extractionis [15].Consequently, semi-organic content
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 ofthedomain)werepreferential.Takingaccountofthesereasons,
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. an=2,mediumconcentration±standarddeviation.
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).
References
[1] P.Vazquez-Roig,R.Segarra,C.Blasco,V.Andreu,Y.Pico,J.Chromatogr.A1217 (2010)2471.
[2]T.Kosjek,E.Heath,TrendsAnal.Chem.30(2011)1065.
[3]Y.Ding,W.Zhang,C.Gu,I.Xagoraraki,H.Li,J.Chromatogr.A1218(2011)10. [4]A.Pal,K.Yew-HoongGin,A.Yu-ChenLin,M.Reinhard,Sci.TotalEnviron.408
(2010)6062.
[5] M.Clara,N.Kreuzinger,B.Strenn,O.Gans,H.Kroiss,WaterRes.39(2005)97. [6]J.P.Besse,J.F.Latour,J.Garric,Environ.Int.39(2012)73.
[7] K.Kummerer,A.Al-Ahmad,Environ.Sci.Pollut.R17(2010)486.
[8]A.C.Johnson,M.D.Murgens,R.J.Williams,K.Kummerer,A.Kortenkamp,J.P. Sumpter,J.Hydrol.348(2008)167.
[9] S.Nussbaumer,P.Bonnabry,J.L.Veuthey,S.Fleury-Souverain,Talanta85(2011) 2265.
[10]O.Zuloaga,P.Navarro,E.Bizkarguenaga,A.Iparraguirre,A.Vallejo,M.Olivares, A.Prieto,Anal.Chim.Acta736(2012)7.
[11] T.A.Ternes,M.Bonerz,N.Herrmann,D.Loffler,E.Keller,B.BagoLacida,A.C. Alder,J.Chromatogr.A1067(2005)213.
[12]H.Runnqvist,S.A.Bak,M.Hansen,B.Styrishave,B.Halling-Sorensen,E. Bjork-lund,J.Chromatogr.A1217(2010)2447.
[13] A.Nieto,F.Borrull,R.M.Marce,E.Pocurull,J.Chromatogr.A1216(2009)5619. [14]T.Okuda,N.Yamashita,H.Tanaka,H.Matsukawa,K.Tanabe,Envrion.Int.35
(2009)815.
[15]Dionex,ASE350TechnicalNote,Doc.065220.
[16] AFNORNFEN872,Qualitédel’eau– Dosagedesmatièresensuspension– Méthodeparfiltrationsurfiltreenfibresdeverre,2005.
[17]J.Seira,C.Joannis-Cassan,C.Albasi,M.Montrejaud-Vignoles,C.Sablayrolles, Int.J.Environ.Anal.Chem.,submittedforpublication.
[18]V.Gabet-Giraud,C.Miege,B.Herbreteau,G.Hernandez-Raquet,M.Coquery, Anal.Bioanal.Chem.396(2010)1841.
[19]A.Nieto,F.Borrull,E.Pocurull,R.M.Marcé,J.Sep.Sci.30(2007)979. [20]A.Jelic,M.Petrovic,D.Barcelo,Talanta80(2009)363.
[21]J.Radjenovic,A.Jelic,M.Petrovic,D.Barcelo,Anal.Bioanal.Chem.393(2009) 1685.
[22]L.Barron,J.Tobin,B.Paull,J.Environ.Monit.10(2008)353.
[23]M.S.Diaz-Cruz,M.J.LopezdeAlda,D.Barcelo,J.Chromatogr.A1130(2006)72. [24]A.Nieto,F.Borrull,E.Pocurull,R.M.Marcé,TrendsAnal.Chem.29(2010)752. [25] A.Saleh,E.Larsson,Y.Yamini,J.A.Jonsson,J.Chromatogr.A1218(2011)1331. [26] J.-B.Baugros,C.Cren-Olivé,B.Giroud,J.-Y.Gauvrit,P.Lantéri,M.-F.
Grenier-Loustalot,J.Chromatogr.A1216(2009)4941.
[27]M.D.Burford,S.B.Hawthorne,D.J.Miller,Anal.Chem.65(1993)1497. [28] B.Halling-Sorensen,S.N.Nielsen,P.F.Lanzky,F.Ingerslev,H.C.H.Lutzhoft,S.E.
Jorgensen,Chemosphere36(1998)357.
[29]T.Steger-Hartmann,K.Kummerer,J.Schecker,J.Chromatogr.A726(1996)179. [30]K.Kummerer,T.Steger-Hartmann,M.Meyer,WaterRes.31(1997)2705. [31] I.Buerge,H.-R.Buser,T.Poiger,M.D.Muller,Environ.Sci.Technol.40(2006)
767.
[32]N.Llewelynn,P.Lloyd,M.D.Jurgens,A.C.Johnson,J.Chromatogr.A1218(2011) 8519.
[33]A.Tauxe-Wursch,L.F.DeAlencastro,D.Granjean,J.Tardellas,Int.J.Environ. Anal.Chem.86(2006)473.
[34]M.Laven,T.Alsberg,Y.Yu,M.Adolfsson-Erici,H.Sun,J.Chromatogr.A1216 (2009)4.
