SusanneNussbaumera,b, PascalBonnabrya,b,Jean-LucVeutheyb,SandrineFleury-Souveraina,∗
aPharmacy,GenevaUniversityHospitals(HUG),Geneva,Switzerland
bSchoolofPharmaceuticalSciences,UniversityofGeneva,UniversityofLausanne,1211Geneva,Switzerland
a r t i c l e i n f o
Articlehistory:
Received11May2011
Receivedinrevisedform15August2011 Accepted16August2011
Available online 24 August 2011
Keywords:
Anticancerdruganalysis Cytotoxicagent
Pharmaceuticalformulation Biologicalsample Environmentalsample Review
a b s t r a c t
Inthelastdecades,thenumberofpatientsreceivingchemotherapyhasconsiderablyincreased.Given thetoxicityofcytotoxicagentstohumans(notonlyforpatientsbutalsoforhealthcareprofessionals), thedevelopmentofreliableanalyticalmethodstoanalysethesecompoundsbecamenecessary.From thediscoveryofnewsubstancestopatientadministration,allpharmaceuticalfieldsareconcernedwith theanalysisofcytotoxicdrugs.Inthisreview,theuseofmethodstoanalysecytotoxicagentsinvarious matrices,suchaspharmaceuticalformulationsandbiologicalandenvironmentalsamples,isdiscussed.
Thus,anoverviewofreportedanalyticalmethodsforthedeterminationofthemostcommonlyused anticancerdrugsisgiven.
© 2011 Elsevier B.V. All rights reserved.
Contents
1. Introduction..........................................................................................................................................2266 2. Analysisofcytotoxicdrugs:generality..............................................................................................................2266 2.1. Analysisofcytotoxicagentsinpharmaceuticalformulations...............................................................................2266 2.1.1. Qualitycontrolofbulkandformulations..........................................................................................2266 2.1.2. Qualitycontrolofpreparedformulationbeforepatientadministration..........................................................2267 2.1.3. Formulationstudies................................................................................................................2267 2.2. Analysisofcytotoxicagentsinbiologicalsamples ...........................................................................................2267 2.2.1. Developmentofnewdrugsandformulations.....................................................................................2267 2.2.2. Therapeuticdrugmonitoring .......................................................................................................2267 2.2.3. Biomonitoringofexposedhealthcareprofessionals...............................................................................2268 2.3. Analysisofcytotoxicagentsinenvironmentalsamples.....................................................................................2268 2.3.1. Surfaceandaircontamination.....................................................................................................2268 2.3.2. Wastewater.........................................................................................................................2268 3. Overviewofanalyticalmethodsforspecificcytotoxicdrugs.......................................................................................2268 3.1. Antimetabolites..............................................................................................................................2268 3.1.1. Pyrimidineanalogues ...............................................................................................................2268 3.1.2. Purineanalogues...................................................................................................................2272 3.1.3. Otherantimetabolites..............................................................................................................2272 3.2. DNAinteractiveagents.......................................................................................................................2273 3.2.1. Alkylatingagents(dacarbazine,temozolomide,procarbazine,ecteinascidin-743)...............................................2273 3.2.2. Cross-linkingagents................................................................................................................2276 3.2.3. Intercalatingagents .................................................................................................................2280 3.2.4. Topoisomeraseinhibitors..........................................................................................................2281 3.2.5. DNAcleavingagents(bleomycin)..................................................................................................2282
∗Correspondingauthorat:Pharmacy,GenevaUniversityHospitals,RueGabrielle-Perret-Gentil4,1211Genève14,Switzerland.Tel.:+41223823978;
fax:+41223823965.
E-mailaddress:sandrine.fleury.souverain@hcuge.ch(S.Fleury-Souverain).
0039-9140/$–seefrontmatter© 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2011.08.034
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2266 S.Nussbaumeretal./Talanta85 (2011) 2265–2289
Cancerisadiseaseinwhichthecontrolofgrowthislostinone ormorecells,leadingeithertoa solidmassofcellsknownasa tumourortoaliquidcancer(i.e.blood orbone marrow-related cancer).Itis oneoftheleadingcausesofdeaththroughoutthe world,inwhichthemaintreatmentsinvolvesurgery, chemother-apy,and/orradiotherapy [1].Chemotherapyinvolvestheuseof low-molecular-weightdrugs toselectivelydestroy tumourcells oratleastlimittheirproliferation.Disadvantagesofmany cyto-toxic agents include bonemarrow suppression,gastrointestinal tractlesions,hairloss,nausea,andthedevelopmentofclinical resis-tance.Thesesideeffectsoccurbecausecytotoxicagentsactonboth tumourcellsandhealthycells[2].Theuseofchemotherapybegan inthe1940swithnitrogenmustards,whichareextremely power-fulalkylatingagents,andantimetabolites.Sincetheearlysuccess oftheseinitialtreatments,alargenumberofadditionalanticancer drugshavebeendeveloped[1].
Anticancerdrugscanbeclassifiedaccordingtotheirmechanism ofaction,suchasDNA-interactiveagents, antimetabolites, anti-tubulinagents,moleculartargetingagents,hormones,monoclonal antibodiesandotherbiologicalagents[2].Inthisreview,themost commonlyusedanticancerdrugs(i.e.classicalcytotoxicagents)are discussed.
-Antimetabolitesareoneoftheoldestfamiliesofanticancerdrugs whose mechanism of action is based onthe interactionwith essentialbiosynthesispathways.Structuralanaloguesof pyrim-idineorpurineareincorporatedintocellcomponentstodisrupt thesynthesisofnucleicacids.5-Fluorouracilandmercaptopurine aretypicalpyrimidineandpurineanalogues,respectively.Other antimetabolites,suchasmethotrexate,interferewithessential enzymaticprocessesofmetabolism.
-DNAinteractiveagents constituteone of thelargestand most importantanticancerdrugfamilies,actingthroughavarietyof mechanisms:
•AlkylatingagentsleadtothealkylationofDNAbasesineither theminorormajorgrooves.Forexample:dacarbazine, procar-bazineandtemozolomide.
•Cross-linkingagentsfunctionbybindingtoDNAresultingto anintra-strandorinter-strandcross-linkingofDNA.Platinum complexes(e.g.,cisplatin,carboplatin,oxaliplatin)and nitro-genmustards(e.g.,cyclophosphamide,ifosfamide)arethetwo maingroupsofthisanticancerdrugsub-family.Nitrosurea com-pounds,busulfanandthiotepaarealsocross-linkingagents.
•Intercalatingagents act bybinding betweenbasepairs. The family include anthracyclines (e.g., doxorubicin, epirubicin), mitoxantroneandactinomycin-D.
•Topoisomerase inhibitors include irinotecan and etoposide compounds.Thesedrugsinhibittheresponsibleenzymesfor thecleavage,annealing,andtopologicalstateofDNA.
•DNA-cleavingagentssuchasbleomycininteractwithDNAand causestrandscissionatthebindingsite.
-Antitubulin agentsinterferewith microtubule dynamics(i.e., spindleformationordisassembly),blockdivisionofthenucleus andleadtocelldeath.Themainmembersofthisfamilyinclude taxanesandvincaalkaloids[2].
Today,withtheincreaseincancerincidence,treatments con-taining cytotoxicdrugs arewidelyused. Duetothe aging(and increasinglycancer-susceptible)populationandthearrivalofnew treatments,thedemandforpharmacycancerservicesisexpected tomorethandoubleoverthenext10years[3].Evenifmore selec-tivetherapiesaredeveloped(e.g.,antibodiesormoleculartargeting agents),treatmentschemeswillcontinue tobeassociated with classicalcytotoxicagents.
Consequently,the needfor analytical methodsto determine anticancerdrugs is of outmostimportance. Thefirst developed methods forthe analysisof cytotoxiccompoundsare based on the use of liquid chromatography with UV detection (LC-UV).
Thesemethodsexhibitedsatisfactoryquantitativeperformancefor theanalysisofsamplescontaininghighconcentrationsoftarget drugs(i.e.developmentofpharmaceuticalformulations,stability studies...).However, in the case of samples withlow amount ofcytotoxics(i.e.biologicalorenvironmentalanalysis),asample preparationstepallowingapre-concentrationoftargetcompounds had tobe appliedbeforetheLC-UV analysis.In the 1990s,the highselectivityandsensitivityofmassspectrometry revolution-izedthewholeanalyticalprocedurebysimplifyingandreducing thesamplepreparationstep.Today,LC–MSisundoubtedlyoneof thetechniquesofchoicefortheanalysisofanticancerdrugswith veryattractive analyticalperformance. Limitof detection(LOD) intheorderofngmL−1arefrequentlyobtained.Otherdetection systemswerecoupledtoLCsuchasfluorimetry,evaporativelight scatteringdetector(ELSD)orelectrochemicaldetection(ECD). Fur-thermore,analyticaltechniqueswerealsopublishedtodetermine anticancerdrugssuchascapillaryelectrophoresiscoupledto UV-detection (CE-UV), amperometric detection or to laser-induced fluorescence (CE-LIF), gas chromatography–mass spectrometry (GC–MS),Ramanspectroscopy,infraredspectrometry(IR).
In thefirstpartof this paper,the needfor analytical meth-odsallowingthedeterminationofthesecytotoxicdrugsinvarious media,suchaspharmaceuticalformulations,biological matrices andenvironmentalsamples,isdiscussed.Inthesecondpart,an overviewofthedifferentanalyticalmethodsisgivenaccordingto specificcytotoxicagents.
2. Analysisofcytotoxicdrugs:generality
2.1. Analysisofcytotoxicagentsinpharmaceuticalformulations Fromtheproductionofcytotoxicbulkuntilchemotherapyin apatient,analyticalmethodsarenecessaryfor(i)qualitycontrol ofbulk and commercialisedformulations,(ii)quality controlof dilutedformulationsbeforepatientadministrationand(iii)studies onformulationsregardingcompatibilityandstability.
2.1.1. Qualitycontrolofbulkandformulations
Forbulkandpharmaceuticalformulations,avaluablemethod forqualitycontrolshouldbeabletosimultaneouslydeterminethe parentdruganditsimpuritiesanddegradationproducts.Quality control,valuable forallpharmaceuticals, mustbein agreement withpharmaceuticalregulations.Usually,separationtechniques offeringgreatselectivity,suchasLCorCE,areused.Amongthe mostcommonlyused detectionsystems, MScanbeconsidered
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thetechniqueofchoice.Itshighselectivityandsensitivityallows thedetectionofverylowconcentrationsofimpuritiesor degrada-tionproducts.Forexample,Jerremalmetal.studiedthestability of oxaliplatin in thepresence of chloride and identified a new transformation product(monochloro–monooxalatocomplex)by LC–MS/MS[4].However,UVspectrophotometrycoupledtoa sepa-rationtechniqueisusedroutinely,butthesensitivityofthemethod mustbesufficientfordegradationorimpurityprofilestudies.For example,MallikarjunaRaoetal.developedastability-indicating LC-UVmethodfordeterminationofdocetaxelinpharmaceutical formulations[5].LC-UVwasalsousedinstudiesofthechemical stabilityofteniposide[6]andetoposide[7]indifferent formula-tions.
2.1.2. Qualitycontrolofpreparedformulationbeforepatient administration
Beforeadministrationtothepatient,commercialised formula-tionsintheformoffreeze-driedpowderorhighconcentrations ofdrug,aredissolvedand/ordilutedwithsodiumchloride(NaCl, 0.9%)orglucose(5%)toobtainthefinalindividualisedquantityof drugprescribedbyaphysicianinanappropriate concentration.
Stabilityofthesedilutedcytotoxicformulationsisoftenlimited(or unknown),andtheyaremostoftenpreparedashorttimebefore patientadministrationbyanurseinthecareunitorinaspecialised unitatthehospitalpharmacy.Evenifpharmaceuticalregulations do not require a final control of each individualised cytotoxic preparation,analysiscanbeappliedtoensurecorrectdrug concen-trationandtoreducemedicationerrorsandtheirconsequencesfor patientswithincreasedriskofmorbidityandmortality[8].
Differentstrategies,usuallyappliedbythehospitalpharmacy, are used to control the prepared formulation before patient administration.Inmostcases,thesemethodsallowapproximate information ontheconcentration tobeobtainedand the cyto-toxicsubstancecontainedinthereconstitutedformulationtobe identified.Giventhehighnumberofcytotoxicpreparationsper dayand the veryshort time betweenprescription, preparation andadministration,simple and fasttechniquesare usually pre-ferredtoconventionalmethods,whichareoftenmoreexpensive andlesseasy-to-handle.Oneapproachconsistsofflowinjection analysis(FIA)withUV-diodearraydetection(DAD).Asshownby Delmasetal.,80%ofcytotoxicpreparations(correspondingto21 differentcytotoxicdrugs)weresuccessfullydeterminedina cen-tralisedpreparationunitinlessthan3.5min[8].However,dueto theabsenceofseparationbeforedetection,thepresenceof excip-ientsintheformulationcaninterferewithFIA-UV/DADanalysis, andcompoundswithsimilarstructurescannotbedistinguished.
Quality control of cytotoxic drugs was also performed by couplingFouriertransforminfrared(FTIR)spectroscopy andUV spectrophotometry [9,10], which increased the selectivity of the method in comparison to single UV. Identification of the drug compound, excipients and drug concentration was thus achieved in a short analysis time withoutsample preparation.
AsforFIA-UV/DAD,additivesincytotoxicformulationsor cross-contamination in the analytical system can perturb analyses.
Moreover,totheauthor’sknowledge,includingquantitative per-formancewithcompletevalidationforqualitycontrolofcytotoxic agentshasnotyetbeendescribedwiththisapproach.
Another, more selective technique for quality control of cytotoxicformulationsmightbeRamanspectroscopy.Itisa non-destructive and rapid method for identifying and quantifying activedrugsandexcipientsinpharmaceuticalformulations[11,12].
Additionally,thisanalysisispossiblewithoutsampling,providing excellentprotectionfortechnicians.AsfortheFTIRandUV/DAD techniques,totheauthor’sknowledge,informationon quantita-tiveperformanceforRamanincytotoxicformulationshasnotyet beenreportedintheliterature.
In conclusion, whenestablishingquality controlof cytotoxic drugs in a daily routine before patient administration, generic FIA-UV/DADassays,FTIRandUV/DADtechniquesorRaman spec-trometry present interesting approaches in terms of time and simplicity.Nevertheless, thelackof selectivityand quantitative dataarethemaindrawbacksofthesetechniques.
2.1.3. Formulationstudies
Variousstudieshavebeenperformedontheattributesof cyto-toxicdrugscontainedinformulations,includingcompatibilityor stability.Thecompatibilityofcytotoxicdrugswithcontainer mate-rialsisveryimportanttoavoidadsorptionordegradationofthe active compound, which both have negative consequences for patienttreatment[13].Inthe1980s,stabilitydataofantitumor agentsinglassandplasticcontainers[14]orintotallyimplanted drugdeliverysystems[15]wereestablished,andareviewof sta-bilitydataforcytotoxicagentswaspublishedin1992[16].Inthese studies,LC-UVwasthemostcommonlyusedanalyticaltechnique.
For new compounds and formulations, stability-indicating methodsallowingseparationofactivecompoundsanddegradation productsarerequiredtoestablishconservationguidelinesforeach cytotoxicdrugindifferentcontainers.InthereviewofBenizrietal., severalstabilitystudieswereevaluated,antineoplasticagentswith sufficientchemicalandphysicalstabilitywereselectedfor home-basedtherapy,andastandardisationofanticancerdrugstability datawasproposed[17].
2.2. Analysisofcytotoxicagentsinbiologicalsamples
Mostofthereportedmethodswereintendedforcytotoxicdrug quantificationinbiologicalmatrices,fundamentalstudiesofnew drugs,pharmacokinetic(PK)andpharmacodynamic(PD)studies, therapeuticdrugmonitoring(TDM)orbiomonitoringfor occupa-tionalexposure.
2.2.1. Developmentofnewdrugsandformulations
The interactionbetweendrugs and DNA is amongthe most important aspects of biological studies in drug discovery and pharmaceutical development processes. A review on differ-ent techniques used tostudy anticancer drug–DNA interaction has been published and included the following techniques:
DNA-footprinting,nuclearmagneticresonance(NMR),MS, spec-trophotometricmethods,FTIRandRamanspectroscopy,molecular modellingtechniques,andCE[18].Furthermore,electrochemical approachescanprovidenewinsightintorationaldrugdesignand would lead tofurtherunderstanding ofthe interaction mecha-nismbetweenanticancerdrugsandDNA[18].PKandPDstudies werefrequentlythereasonforthedevelopmentofnew analyti-calmethodstodeterminecytotoxicagentsinbiologicalsamples (e.g.,urine,serum,plasma,intracellularmatrix,tissues).For exam-ple,arecentlyreportedLC–MS/MSmethodfordocetaxelinplasma wasfoundtohavebetterperformancethanpreviouslyreported methodsintermsofsensitivity,anditappearedtobeapromising methodforalargeclinicalpharmacologystudy[19].
2.2.2. Therapeuticdrugmonitoring
TDMforchemotherapyagentsisnotcurrentlyusedroutinely, mainly due to the lack of established therapeutic concentra-tion ranges. Combinations of different chemotherapies make the identification of a target concentration difficult, as the concentration–effectrelationshipdependsonthedifferent treat-ments [20]. However, TDM has the potential to improve the clinicaluseofsomedrugsand toreducetheseveresideeffects ofchemotherapy.Forexample,Rousseauetal.reporteddifferent possibilitiesandrequirementsforTDM[21].Mostcommonly,TDM isperformedfor methotrexate[2].Reviewsondrugmonitoring
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werealreadypublishedin1985byEksborgandEhrsson[22],and hyphenatedtechniquesinanticancerdrugmonitoring(e.g.,GC–MS, LC–MSandCE-MS)werepublishedbyGuetensetal.in2002[23,24].
2.2.3. Biomonitoringofexposedhealthcareprofessionals
Cytotoxicdrugshavebeenrecognisedashazardousfor health-careprofessionalssincethe1970s[25],anddifferentstudieshave shownhowoccupationalexposuretoantineoplasticdrugsis asso-ciated witha potential cancer risk [26–29]. However, a direct relationship betweenexposure to cytotoxic contamination and harmfuleffectsisdifficulttoestablish,andnomaximalacceptable amountforthesedrugshasbeensetbyregulationofficesuntilnow.
Biomonitoringrequiresverysensitiveandselectivemethodsfor traceanalysisofcytotoxicdrugsinurineorbloodsamples. More-over,validatedandstandardisedmethodsarelackingforcytotoxic agentmonitoringinbiologicalsamplesofhealthcareprofessionals [30,31].Theconcentrationofcytotoxicdrugsinbiologicalsamples fromhealthcareprofessionals,whichareexposedtothese com-pounds,isusuallylowerthanforbiologicalsamplesfrompatients receivingformulationswithdrugamountintheorderofmg.Even ifdruglevelsareusuallylowerinurinethaninbloodsamples,urine samplesarepreferredforpracticalreasons.Thatiswhymethods usedfortheanalysisofcytotoxicdruginsamplesof healthcare professionalshavetoexhibitasufficientsensibilitytoallow reli-ablequantificationofthesecompounds.GC–MSand LC–MSare themostcommonlyused[32,33],butaccordingtotheanalytes, othertechniquesmayalsobeinteresting(forexample,inductively coupledplasma-massspectrometry(ICP-MS)orvoltammetryfor platinumcompounds[34,35]).Mostreportedstudieshavefound cytotoxicdrugsintheurineorbloodofhealthcareprofessionals despitesafetystandardsforhandlingthesecompounds[36–39].
Accordingtoprecautionaryprinciples,exposureshouldtherefore bekepttothelowestpossiblelevels[40].
2.3. Analysisofcytotoxicagentsinenvironmentalsamples 2.3.1. Surfaceandaircontamination
Acompletereviewofanalyticalmethodsusedfor environmen-tal monitoringof antineoplastic agents was published in 2003 by Turci et al. [36]. Analytical methods for the quantification ofone ortwo model cytotoxicagents andgeneric methods for thedetermination ofseveraldrugshavebeendeveloped.When usingmarkercompounds,wipe sampleshave beenobtainedby compound-specificwipingproceduresfollowedbyadapted ana-lytical techniques (e.g., voltammetry for platinum drugs [41]).
Suchmethodsformarkercompoundspresentedverygood quan-titative performance regarding detection limits and estimated potentialsurfacecontamination[41–46].However,awiderange ofchemotherapyformulationswithdifferentdrugsanddifferent preparation procedures are usually produced in hospital units.
Therefore, to get an overview of several contaminants, multi-compoundmethodsarerequiredwithgenericwipingprocedures.
Forsufficientselectivityandsensitivity,LC–MS/MSisoneofthe analyticalapproachesofchoice[47–53].
2.3.2. Wastewater
Afteradministrationofanticancerdrugstopatients, consider-ableamountsofcytotoxicagentsareeliminatedintheurineand therebyreachthewastewatersystem.Duetotheirpotential toxic-itytohumansandtheenvironment,analysisofcytotoxicdrugsand theirmetabolitesisalsoneededinhospitaleffluentsand wastew-atersamples.Variousanalyticaltechniquescanbeusedfor this purpose,includingICP-MSforplatinumcompounds[54],CE-UVfor fluorouracil[55],LCwithfluorescencedetectionforanthracyclines
[56]andLC–MS/MSforantimetabolites[57]andothercytotoxic agents[58,59].
3. Overviewofanalyticalmethodsforspecificcytotoxic drugs
InthisSection,analyticalmethodsforeachcytotoxicdrugare discussed.Only themost commonlyused cytotoxicagents, i.e., antimetabolites,DNAinteractiveagentsandantitubulinagents,are consideredinthispaper.
3.1. Antimetabolites
Analysisofpyrimidineanalogues,purineanaloguesandother antimetabolitesaredescribedinthissection.Thechemical struc-tures of antimetabolites are shown in Fig. 1, and published analyticalmethodsfordeterminationofthesecompoundsin phar-maceuticalformulations,biologicalandenvironmentalsamplesare reportedinTable1.
3.1.1. Pyrimidineanalogues
3.1.1.1. 5-Fluorouracil,tegafur,capecitabine. 5-Fluorouracil(5-FU) isawidelyusedcytotoxicagentforthetreatmentofbreasttumours andcancersofthegastrointestinaltract,includingadvanced col-orectalcancer.Itisalsoeffectiveforcertainskincancersbytopical administration.Themain sideeffects include myelosuppression andmucositis[2].Tegafurandcapecitabinearemetabolisedto5-FU andaregivenorallyformetastaticcolorectalcancer.
Fewstability-indicatingLC-UVmethodsforstabilitystudiesof 5-FUinpharmaceuticaldosageformscontainingvariousadditives [60,61]and inratcaecaltissues[62]have beendeveloped with goodquantitativeperformanceintermsofaccuracyandprecision.
Simplesamplepreparationincludingcentrifugationanddilution wasperformedand anLOQof500ngmL−1 wasachievedfor 5-FUinratcaecaltissues[62].However,5-FUwasobservedtobe degradedunderalkalineconditions,whileonlynegligible degra-dationwasobservedinacidic,neutral, oxidativeand photolytic conditions.Drugcombinationsof5-FUanddoxorubicinwerealso successfullydeterminedbyLC-UVininjectionsolutionsand bio-logicalsamples[63].Acompleteseparationbetweendoxorubicin andmethylhydroxybenzoate,usedasapreservative,wasobtained.
Generally,publishedmethods fortheanalysisoftegafurand
Generally,publishedmethods fortheanalysisoftegafurand