ContentslistsavailableatScienceDirect
Journal
of
Pharmaceutical
and
Biomedical
Analysis
jo u r n al h om epag e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a
HPLC-FLD
methods
to
quantify
chloroaluminum
phthalocyanine
in
nanoparticles,
plasma
and
tissue:
application
in
pharmacokinetic
and
biodistribution
studies
Líliam
Teixeira
Oliveira
a,
Giani
Martins
Garcia
a,
Eunice
Kazue
Kano
a,1,
Antônio
Cláudio
Tedesco
b,
Vanessa
Carla
Furtado
Mosqueira
a,∗aCiPharma-ProgramadePós-graduac¸ãoemCiênciasFarmacêuticas,EscoladeFarmácia,UniversidadeFederaldeOuroPreto,RuaCostaSena,171-Centro,OuroPreto35400-000,
MinasGerais,MG,Brazil
bDepartamentodeQuímica,FFCL-RP,UniversidadedeSãoPaulo,Av.Bandeirantes,3900-MonteAlegre,RibeirãoPreto14040-901,SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received22December2010 Receivedinrevisedform12April2011 Accepted16April2011
Available online 22 April 2011 Keywords: Chloroaluminumphthalocyanine High-performanceliquid chromatography-fluorescencedetection Nanocapsules Photodynamictherapy Fluorescentdye
a
b
s
t
r
a
c
t
Analyticalandbioanalyticalmethodsofhigh-performanceliquidchromatographywithfluorescence
detection(HPLC-FLD)weredevelopedandvalidatedforthedeterminationofchloroaluminum
phthalo-cyanineindifferentformulationsofpolymericnanocapsules,plasmaandliversofmice.Plasmaand
homogenizedliversampleswereextractedwithethylacetate,andzincphthalocyaninewasusedas
inter-nalstandard.Theresultsindicatedthatthemethodswerelinearandselectiveforallmatricesstudied.
Analysisofaccuracyandprecisionshowedadequatevalues,withvariationslowerthan10%inbiological
samplesandlowerthan2%inanalyticalsamples.Therecoverieswereashighas96%and99%intheplasma
andlivers,respectively.Thequantificationlimitoftheanalyticalmethodwas1.12ng/ml,andthelimits
ofquantificationofthebioanalyticalmethodwere15ng/mland75ng/gforplasmaandliversamples,
respectively.Thebioanalyticalmethoddevelopedwassensitiveintherangesof15–100ng/mlinplasma
and75–500ng/ginliversamplesandwasappliedtostudiesofbiodistributionandpharmacokineticsof
AlClPc.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Phthalocyanine(Pc)derivativeshaveattractedmuchattention becauseoftheirparticularpropertiesandhavebeenusedas pho-tosensitizersandasfluorescentdyes.Theyhavebeeninvestigated inseveralapplicablematerials,suchaschemical sensors,liquid crystalsand nonlinear optics. They have alsobeen usedin the pharmaceuticalfield asphotosensitizersforphotodynamic ther-apy(PDT)[1–4].PDTisabimodaltherapeuticstrategybasedon aphotosensitizeractivatedbyvisiblelight[2].Thistechniquehas beenproposedasanalternativeorasacomplementtoconventional protocolsinthetreatmentofmalignanttumors[3].Currently,PDT basedonclassicalphotosensitizers,hematoporphyrinorsynthetic porphyrinderivativesisinpreclinicalandclinicaluse[5–8].
PDTbasedonphthalocyanineshasremarkablemedical poten-tial because of improved selectivity and efficiency against tumorcellsand microorganismproliferation[9,10]. Phthalocya-ninesexhibit a highabsorptioncoefficientin thevisibleregion
∗ Correspondingauthor.Tel.:+553135591032;fax:+553135591628. E-mailaddresses:vamosqueira@gmail.com,mosqueira@ef.ufop.br
(V.C.F.Mosqueira).
1 Presentaddress:FaculdadedeCiênciasFarmacêuticasdaUniversidadedeSão
Paulo,Brazil.
of the spectrum, primarily in the phototherapeutic window (600–800nm),andhavealonglifetimeofthetripletexcitedstate inphotosensitizers,whichisnecessarytoproduce1O
2(thetoxic species)efficiently[11].Thesefeatureshaveledtoproposalsfor their development for clinical use. However, some drawbacks related tophthalocyaninesolubilityand bioavailabilitymust be addressedfor ittobesuitablefor clinicaluse,particularlywith chloroaluminumphthalocyanine(AlClPc),whichisvery hydropho-bic[12].AlClPcpresentsoptimalcharacteristicsforuseinPDT,such aslong-livedexcitedsingletandtripletstates[11].Furthermore, despitethegrowingknowledgeinthephotophysicaland photo-chemicalaspectsofAlClPcandinitsmedicalapplications,thereis aslowevolutionintheanalyticalmethodologiesforits determi-nationinbiologicalsamples.Themethodspreviouslyreportedto determineAlClPcconcentrationsinbiologicalsamplesarebasedon spectrophotometricandspectrofluorimetricassays[13,14].Optical quantificationmethodsinbulktissuesorhomogenatesare gener-allyrestrictedintheirdetectionlimitandaccuracy.Thesemethods havenotbeenvalidatedingeneralfortheAlClPcassayinbiological matricestoprovidetherequiredselectivity,sensitivity,precision, accuracy,stabilityandrecovery.Thus,asuitableliquid chromato-graphicmethodthatwillallowanalysisofAlClPcinpre-clinicaland clinicalstudiesisstilllacking.
Additionally, intravenous administration of the hydrophobic AlClPcisgreatlyhamperedbyitslowsolubility.Thus,thisspecific
0731-7085/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.04.016
N N N N N N N N Al Cl N N N N N N N N Zn
Fig.1. ChemicalstructuresofAlClPcandZnPc.
physicochemicalcharacteristicofAlClPcrequiresadvanced tech-nologiestoformulatenanometricdeliverysystems,toimproveits dispersioninwater,enhancethespecificuptakebytargetedtissues andimprovePDTefficiency[15,16].Recently,Siqueira-Mouraet al.describedvalidatedspectrophotometricandspectrofluorimetric methodsforAlClPcquantificationinnanocarriersintherangesof 0.50–3.00and0.05–1.00g/ml,respectively[17].However,AlClPc wasnotdeterminedinbiologicalsamplesandnoextraction pro-cedure was described. A literature survey indicated that there wasnoreportofavalidatedmethodofhigh-performanceliquid chromatographywithfluorescencedetection(HPLC-FLD)forthe determinationofAlClPcinbiologicalsamplesandnanocarriers.
Inthisstudy,asuitableanalyticalmethodforthequantitative analysisofAlClPcinthreedifferentnanocapsuleformulationsas wellasinbiologicalsamplesofmiceplasmaandliverwas devel-opedandvalidatedusingHPLC-FLD.Thetissueconcentrationsof AlClPcintheliversandplasmaofmice20minafterintravenous administrationweremeasured.Furthermore,thepharmacokinetic profileoffreeAlClPcsolutionandAlClPcloadedinPLA-NCwere determinedtoillustratetheapplicationofthemethoddescribed.
2. Experimental
2.1. Chemicals
AlClPc (chloro(29H,31H-phthalocyaninato)aluminum) and ZnPc (zinc(II)phthalocyanine) were purchased from Sigma (Sigma–AldrichCo., St.Louis,MO,USA).Theacetone,methanol, anddimethylformamide usedfortheHPLCanalyseswereHPLC gradeandwerepurchasedfromTedia(Brazil).Milli-Qwaterwas purifiedusingaSymplicity®System(Millipore,Bedford,USA)and wasusedtoprepareallthesolutions.Soylecithin(Epikuron170) wasagiftfromLucasMeyer(France).Thepurifiedmedium-chain triglyceride (MCT) waspurchased from Hulls(Germany). Ethyl acetate,polyethyleneglycol300anddimethylsulfoxide (analyt-icalgrade) were purchased fromVetec (Rio de Janeiro, Brazil). PLA [poly(d,l-lactide), Mw 75,000–120,000g/mol], low molec-ular weight chitosan (CS) and Poloxamer 188 were purchased from Sigma–Aldrich (Brazil). PLA-PEG [poly(d,l-lactide)-co-polyethylenoglycolaverageMw66,000g/mol]withaPEGblock ofMn5000g/molwasagiftfromAlkermes(Cambridge,USA)and wasusedwithoutfurtherpurification.
2.2. Nanocapsulespreparation
AlClPc loaded-nanocapsules, 0.1mg/ml of PLA and PLA-PEG werepreparedaccordingtoFessietal.[18]andMosqueiraetal.
[19],respectively.Briefly,thepolymer(0.6%,w/v),oil(0.75%,w/v),
soy lecithin(0.75%,w/v) andAlClPc (0.1mg/ml)were dissolved inacetone(15ml).Theorganicphasewaspouredintothe aque-ous phase (30ml) containing poloxamer 188 (0.75%,w/v), and thesolutionwasstirredfor10min.Thesolventswereevaporated underreducedpressureupto10ml(HeidolphRotaryEvaporator, Germany).PLA-CSnanocapsules(NC)werepreparedbythesame procedureabove,replacingpoloxamerbythechitosan(0.25%,w/v) intheaqueousphase.Thesizeoftheparticleswasdeterminedby photoncorrelationspectroscopy(PCS)(NanosizerN5 Plus Beck-mannCounter,USA)afteranappropriatedilutionofthesamplesin ultra-pureMilli-Qwater.
2.3. Calibrationstandards
Astocksolution(100g/ml)ofAlClPcwaspreparedinethanol, andZnPcwasusedasinternalstandard(I.S.)becauseofits solubil-ityprofileinethanol,itschemicalstabilityandchemicalsimilarity toAlClPc(Fig.1).Furthermore,itwaspreviouslyusedasanI.S.in quantitativeassaysinbiologicalsamples[20].Stocksolutionsof AlClPcandZnPc(I.S.)inethanolatconcentrationsof1g/mland 5g/ml,respectively,werepreparedasstandardsandquality con-trolsamples(QC)forAlClPcquantificationinbiologicalmatrices. CalibrationcurveswereconstructedusingAlClPcconcentrationsof 150ng/ml,200ng/ml,400ng/ml,500ng/ml,800ng/ml,900ng/ml and1000ng/ml(workingsolutions).Theworkingsolutionswere preparedviatheserialdilutionmethodfromthestocksolutionand allsolutionswerestoredat−20◦C.
2.4. Chromatographicconditions
The HPLC system employed consisted of a Waters Alliance 2695 separation module composed of an autosampler, pump, columnovenandfluorescencedetector(Waters2475).The sep-aration was performed on a 150mm×4.6mm, 4-m particle size C18 Gemini Phenomenex column protected by a Phe-nomemexsecurityguardAJO-7597C18column(2mm×4.6mm, 3m). The isocratic mobile phase consisted of a mixture of methanol/acetone/dimethylformamide(90:5:15,v/v/v).Itwas pre-pareddaily, degassedbeforeuseand pumped at1ml/min. The analysiswasperformedat30◦C.Theinjectionvolumewas30l forbiologicalsamplesand10lfortheothersamples.Thecolumn eluentwasmonitoredwithafluorescencedetectoratwavelengths of610nmand675nm,forexcitationandemission,respectively.
2.5. Analyticalmethod
Thenanocapsuleswereanalyzedbyananalyticalmethod,which was validated in accordance with International Conference on
prepared by diluting thestock solution (100g/ml) in ethanol toobtainconcentrationsof0.025,0.05,0.1,0.5,1.0,2.0,4.0and 5.0g/ml.Thecalibrationcurvewasconstructedbylinear regres-sion of theplot of AlClPc peak area against thecorresponding concentration. Quality-control samples were prepared at con-centrationsof 0.5, 2.0 and 4.0g/mlAlClPc todeterminatethe precisionandaccuracyoftheanalyticalmethod.Theselectivityin thepresenceofpolymersandexcipientsusedintheNC formula-tionswasinvestigatedbypreparingamobilephasespikedwith drug-freePLA,PLA-PEGandPLA-CSnanocapsules.The nanocap-suleformulationswerecompletelydisruptedanddissolvedinthe mobilephasebeforethechromatographicanalysis.
ThequantificationofAlClPcinthepolymericnanocapsulesof PLA,PLA-PEGandPLA-CSwasperformedasdescribedbelow.The percentageofencapsulation(%drugloading)ofAlClPcin nanocap-suleswascalculatedasthedifferencebetweenthetotaldruginthe finalcolloidalsuspensionandthefreedrugintheexternalaqueous phase.TheAlClPcnotencapsulatedcanbefounddissolvedinthe externalaqueousphaseorprecipitated.Todeterminetheamount ofdrugthatprecipitatedinthecolloidalNCsuspension(not encap-sulated),thesolutionwasfilteredusinga0.8mfiltertoremove AlClPc crystals.Thefraction of AlClPcdissolved inthe external phase(ultrafiltrate)wasassessedbycentrifuging400lsamples ofNC(afterfiltration)in anAMICOMdevice(Microcon Ultrafil-ter,MWCO50,000Da,Millipore®)at500×gfor15min.TheAlClPc associatedwiththeNCwasretainedintheuppercompartmentof thedevice.
TheamountofAlClPcloadedontheNCwasdeterminedfrom Eq.(1)andtheencapsulationefficiencyofAlClPcintotheNCwas determinedbyEq.(2),whereA=concentration:
(%) = A
total−Aultrafiltrate
Atotal ×100 (1)
(%) = Atotal−Aultrafiltrate
Aweighed ×100 (2) FortotalAlClPcdeterminationintheNC,100loftheNC sus-pension wasaddedto 2.5ml ofacetonitrile,vortexedfor 5min (VortexInstrument,IKA,Germany),andcentrifuged;25lofthe supernatant wasthen injectedinto theHPLC system.Thisstep disruptedthenanocapsulesandreleasedthedrug.For quantifi-cationoftheunencapsulateddrug,50loftheultrafiltratewas mixedwith200lacetonitrile.Themixturewascentrifugedand thesupernatantassayedusingtheHPLC.Allsamplesanalyzedby HPLCwerefilteredusinga0.45mfilter(4mm)beforeinjection (25l)intotheHPLCsystem.Theanalysiswasperformedin tripli-cate.
2.6. Animals
Thein vivo experimentswere approved bythe Ethics Com-mitteeonAnimal Experimentation of the UniversidadeFederal deOuroPreto,Brazil(protocolno.2009/29)and arein compli-ancewiththeGuidefortheCareandUseofLaboratoryAnimals recommendedbytheInstituteofLaboratoryAnimalsResources
[22].FemaleSwissmicewereusedinthisstudy.Theyweighed 23.5–24.5gandweremaintainedatthefollowingenvironmental conditions:12hday/nightcycle,temperature22±2◦C,standard dietandwateradlibitum.
2.7. Biologicalsamplepreparation
Plasmaandliversamplesfrom drug-freeanimalswereused asblanks for method validation. Bloodfromanesthetized mice (ketamine 60mg/kg and xylazine 7.5mg/kg) was collected in
separatedby centrifugationat 400×gfor 10minand wasthen aliquotedandstoredat−80◦Cuntilanalyzed.Afteranimalswere euthanized,thetissueswereremoved,washedwithcoldsalineand blottedontofilterpaper.Piecesofliver(200mg)werecut,weighed andstoredat−80◦Cuntilneeded.
For the quantification of AlClPc in plasma, plasma samples (80l)wereplacedintoEppendorfmicrocentrifugetubes;AlClPc standards(10l)ofeachworkingsolutionand10lofI.S.(ZnPc 5g/ml)wereaddedtoeach tube.Thesolutionsweremixedin avortexandextractedwith1mlofethylacetate.Then,the sam-pleswereshakenonavortexfor10minatthesamespeedforall samples.Afterwards,tubeswerecentrifugedat9300×gfor10min andtheresultingupperorganiclayerswerepooled.Foreach sam-ple,this procedurewasrepeated3timesand theorganiclayers werepooled,filteredusing0.45-mfilters (Millipore,USA)and evaporatedtodrynessinanitrogen-stream.Aftertheextraction procedure,eachsamplewasreconstitutedin100lofthemobile phase.
For quantification in the liver samples, thawed pieces liver (200mg)werehomogenizedfor1mininaround-bottomplastic tubewith1mlsodiumphosphatebuffer(pH6.5)atroom temper-atureinanultrasonicprocessor(VibracellTMVC750)withametal rodtitaniumprobeat300W.Thetissuehomogenateswerestored at−80◦Cuntiltheextraction.AlClPcwasextractedfrom80lof thehomogenatetissueusingthesameproceduredescribedabove forplasmasamples.
2.8. Assayperformance
The method was validated according to FDA guidelines for validationofbioanalytical methods[23].Theassayperformance wasevaluatedthroughthedeterminationofselectivity,limitsof detection(LOD),limitsofquantification(LOQ),thelinearityover thetestedconcentrationrange(from15 to100ng/mland 75to 500ng/gformiceplasmaandliversamples,respectively),recovery, accuracy,precision,andthestabilityofthebioanalyticalmethod. Because ofthelight-absorbing propertiesof AlClPc,all samples wereprotectedfromlight;preparationsandextractionswere per-formedindarkenedrooms.
2.8.1. Selectivity
Theselectivityofabioanalyticalmethodisgenerallydefinedas thelackofinterferingpeaksattheretentiontimesoftheassayed drugand theinternalstandard inthechromatograms[23].The selectivityoftheassaywasinvestigatedbyprocessingand ana-lyzingblankspreparedfromsixindependentlotsofplasmaand liversamplescontrols.
2.8.2. Limitofdetectionandlimitofquantification
Thelimitofdetection(LOD)ofthebioanalyticalmethodswas definedasthelowestconcentrationlevelgivingapeakareaofthree timesthenoise.TheLOQwasdeterminedasthelowest concentra-tiononthestandardcalibrationcurve,whichcanbequantitatively determinedwithaprecisionof20%andaccuracyof80–120%[23].
2.8.3. Linearity
Thelinearity of ananalytical procedureis itsability (within a given range) to obtain test results that are directly propor-tional to the concentration (amount) of analyte in the sample
[21]. The linearity of the bioanalytical assay for the test com-poundswasevaluatedwithatotalofsevencalibrationsstandards over the concentration range of 15–100ng/ml for plasma and 75–500ng/gforliver.Calibrationcurveswereconstructedbythe linearleast-squaresregressionanalysisbyplottingthepeak-area ratios(AlClPc/I.S.)versusthedrugconcentrations.Thecalibration
modelwasacceptedif(a)thecoefficientofcorrelationwasgreater thanorequalto0.98and/or(b)residualswerewithin±20%atthe lowerlimitofquantificationandwithin±15%atallother calibra-tionlevels[23].Fivecalibrationcurveswerepreparedfromablank sample(matricessamplesprocessedwiththeinternalstandard), includingtheLOQ.
2.8.4. Recovery
Thetruenessofananalyticalprocedurereferstothecloseness ofagreementbetweenaconventionallyacceptedvalueandamean experimentalvalue.Thetruenesswasexpressedasthepercentage recoveryofthetargetvalueassessedbythevalidationstandards intheplasmaandliveratthreeindependentconcentrationlevels: low,mediumandhigh[23].Recoverywascalculatedbycomparing themeanpeakareaobtainedfromanextractedsample(recovered %)tothepeakareaobtainedafterthedirectinjectionofa solu-tionofthesamedrugconcentrationdilutedinthemobilephase (100%).The%recoverywasdeterminedbycomparingthe concen-trationsofthreequalitycontrolsamples(n=6,20,40and80ng/ml forplasmasamplesand100,200and400ng/gforliversamples) withunextractedreferencestandardscontainingthesameamount ofthe analyte.The sampleswere preparedfrom blankplasma, whichwasextractedthreetimesusingethylacetate,vortexedfor 10minandcentrifugedfor10minat9300×g.Thesupernatants weredrawnandthestandardsolutionsandinternalstandard solu-tionswereaddedtoprovidethereferencestandardsthatweredried andreconstitutedwiththemobilephaseforanalysis.
2.8.5. Accuracyandprecision
Theintra-batch accuracy and precision weredetermined by theanalysisofsixreplicatesoflow(20ng/ml),medium(40ng/ml) andhigh(80ng/ml)concentrationsoftheanalyteinmiceplasma andlow(100ng/g),medium(200ng/g)andhigh(400ng/g) con-centrationsinmiceliver.Inter-batchaccuracyandprecisionwere determinedbytheanalysisofthesequalitycontrol(QC)sampleson threeseparateoccasions.Theoverallprecisionofthemethodwas expressedasrelativestandarddeviation(RSD)andtheaccuracyof themethodwasexpressedintermsofrelativeerror.Theaccuracy andprecisiondeterminedateachconcentrationlevelofthecurve pointsmustbewithin±15%oftherespectivenominalvalue. 2.8.6. Stability
Thedrugstabilityinbiologicalmatricesisafunctionofthe stor-ageconditions,thechemicalpropertiesofthedrugandthematrix
[23].Forallstabilitystudies,low,mediumandhighplasmaquality control(QC)sampleswereusedintriplicate.Thestabilityat24h (aftertheplasmasampleextractshadbeenexposedtothesame temperatureastheautosamplerenvironment)wasdeterminedfor theanalytesandfortheI.S.Thefreeze–thawstabilityofthe sam-pleswasobtainedoverthreefreeze–thawcycles,bythawingthe QCsamplesunassistedatroomtemperatureandthenrefreezing for12–24hfollowedbyanalysis.Thebench-topstabilitywas eval-uatedbykeepingtheQCsamplesatroomtemperaturefor4hand thenthesampleswereprocessedandanalyzed.TheAlClPc stabil-itywasevaluatedat−80◦Cfor30days.TheconcentrationofAlClPc aftereachtestconditionwascomparedtotheinitialconcentration addedtothesample.TheworkingsolutionsofAlClPc(40ng/ml) andtheI.S.(500ng/ml)wereanalyzedimmediatelyafter prepara-tionandafterbeingstoredfor30daysat−20◦C.Subsequently,the concentrations(20,40and80ng/mlinplasmasamplesand100, 200and400ng/ginliversamples)werecompared.
2.9. Applicationofthemethod
To test the validated methods with real samples, experi-ments were undertaken to determineAlClPc in three types of
nanocapsulesof differentchemical constitutionsusing the ana-lyticalmethoddeveloped.Thebioanalyticalmethodwasusedto determineAlClPcconcentrationsinplasmaand liversamplesof healthymice(n=4).Themicereceivedsingleintravenousdosesof 50,100,200or300lof0.1mg/mlAlClPc-loaded,PLA nanocap-sules.Thebloodsamples(approximately500l)werecollected inheparinized tubes20minafteradministration.Sampleswere immediatelycentrifugedfor10minat400×g,andtheplasmawas thenfrozenat−80◦Cuntilanalysis.Aftereuthanasiaofthemice, theliverswereexcised,washedwithcoldsalineandblottedwith filterpaper.ThequantificationofAlClPcinbiologicalsampleswas undertakenwithin12–24hpost-collectionofsamplesusingthe methoddescribedinSection2.7.TheQCsampleswereincorporated intotheseriesonthesamedayoftheanalysis.
ThepharmacokineticstudywasperformedwithfreeAlClPcand AlClPcloadedinPLA-NC.ThefreeAlClPcsolutionwasprepared dis-solving400gofthedrugin400lofDMSOand600lofPEG 300. Afterwardthis solutionwasdiluted in5%glucosesolution upto0.1mg/mlofAlClPcfinalconcentration.TheAlClPcsolution and AlClPc PLA-NC suspension were filtered ona 0.8m ster-ilefilterand150lwereadministeredintravenouslyinmice.At theindicatedtimeintervals,5,20,60minand 3,6,12,24,48h post-injection,blood(200l)wascollectedinheparinizedtubes, centrifugedfor15minat800× gandplasmawascollected.At50l ofplasmasamples10lofI.S.solutionwereadded.Thesamples werethen extractedby themethoddescribed above.To deter-minetheamountoftotalfluorescentdye(AlClPc)inplasma,atotal plasmavolumepermouseof4.9%ofbodyweightwasassumed
[24].Theresultswereexpressedasa percentageoftheinjected doseand are themean±standard errors (n=6 mice). To com-parethedifferentformulations,theareaunderthecurve(AUC) (concentration×time)offreeAlClPcandAlClPcloadedinPLA-NC were calculated by the trapezoidal method during the experi-mentalperiod(AUC[0–48]).Theextrapolatedarea(AUC[t−ω])was calculatedbydividingthelastexperimentalconcentration(Ct)by therateconstantofelimination(K)becausethisarearepresented approximately1%oftheexperimentalAUC,itwasneglectedinour calculations.ThestatisticalanalysiswasperformedusingPrisma® 5.0software.Thedifferenceslowerthanp<0.01wereconsidered significant.Thus,theoverestimatedplasmaclearance(Cl)was cal-culatedfromthefollowingequation:Cl=Doseiv/AUC0–48.
3. Resultsanddiscussion
Using the HPLC-FLDanalytical method developed, AlClPc in nanocapsulesexhibitedaretentiontimeof1.97mininthe experi-mentalconditionsutilizedandthecalibrationplotwaslinearinthe concentrationrange investigated (0.025–5.0g/ml). Theslopes, interceptsandthecoefficientsofdeterminationwerefoundtobe 13979031(a),356534(b)and0.9997(r2),respectively.Thelimitsof detection(LOD)werecalculatedbytheequationLOD=3.3/Sand thelimitofquantification(LOQ)wascalculatedbytheequation LOQ=10/S,whereisthestandarddeviationofy-interceptsof regressionlinesandSistheslopeofthecalibrationcurve.TheLOD andLOQvalueswere1.12ng/mland3.39ng/ml,respectively.The HPLC-FLDmethodshowedmoresensitivitythanthe spectrofluori-metricmethodsdescribedbySiqueira-Mouraetal.whichshowed LODandLOQvaluesof10and40ng/ml,respectively[17].The chro-matogramofAlClPcwasnotalteredinthepresenceofpolymers andotherchemicalconstituentsusedinPLA,PLA-PEGandPLA-CS NCformulations,suchaspolymers,surfactantsandoilsin accor-dancewithpreviouslyobserveddata(Fig.2)[17].Todetermine intra-assayaccuracyandprecision,sixreplicateanalyseswere per-formedateachofthethreeconcentrations(0.5,2.0and4.0g/ml). ThemeansforquantificationofAlClPcwithqualitycontrol
sam-Accuracyandprecisiondeterminedbyanalyticalmethod.
Concentration (g/ml)
Precision(RSD%) Accuracy(%)
Intra-daya Inter-daya Intra-daya Inter-daya
0.5 1.00 1.35 101.43 101.84
2.0 1.80 1.67 99.77 101.88
4.0 0.50 1.48 101.90 100.59
RSD,relativestandarddeviation.
an=6.
plesin thenanocapsulesaresummarized in Table1.Themean intra-dayprecisionvaluesforallsampleswerefoundtobewithin 0.50–1.80%,andtheinter-dayprecisionvalueswerebetween1.35% and1.67%.Theintra-dayaccuracywasfoundtorangefrom99.77 to101.90%and100.59to101.88%fortheinter-dayaccuracy. More-over,therelativestandarddeviation(RSD)obtainedwithin-dayand between-dayexperimentswerebelow2%ineachcase.
During the development and validation of the bioanalytical methodofquantificationforAlClPcinmiceplasmaandliver,five setsofcalibrationstandardswerepreparedforeachmatrix.The retentiontimesfor AlClPcandfor theI.S.atthis pointofstudy were2.036and4.816min,respectively(Fig.2).Theassayswere linearbetween15ng/ml and100ng/mlinplasma and between 75ng/gand500ng/ginliversamples.Themeanregression equa-tionoffive standardcurves was:y=(0.0235)x+(0.0207)forthe plasmaand y=(0.0221)x+(0.1650) forthe liversamples,where yisthepeakarearatioofAlClPc/I.S.andxistheconcentration ofAlClPc.Thecalibrationcurvesgeneratedshowedagood rela-tionshipbetweentheareaofthepeakofAlClPc/I.S.(y)andthe respectiveAlClPcconcentration(x),withr2=0.9873fortheplasma andr2=0.9913fortheliversamples.TheLOQandLODwerefound tobe15ng/mland5ng/mland75ng/gand25ng/g,respectively forplasma and liversamples.Thelimit ofquantification inthe liversamples(LOQ=75ng/g)ofthemethoddevelopedhereinwas
[24].
The selectivity of the method was evaluated by processing drug-freeplasmaandliversamplesobtainedfromsixmice(Swiss, female) in a similar manner to the calibration standards and unknowns.Chromatogramswerecomparedtodetermineifthere wasanyinterferencefromthematrix orfromanyof theassay reagents.Themethodwasselectiveinbothmatrices.Therewere noendogenousinterferencesinplasmaandliverfromthemicein thedeterminationoftheAlClPcandtheI.S.(Fig.2).Toquantify theAlClPcextractedfrombiologicalmatrices,theinherenthigh fluorescencequantumyieldsofthePcswereexploited.Atthis spec-tralrange(600–700nm),theintrinsicbiomolecules,suchasNADH, porphyrinsandcollagendisplayedminimalexcitability,hencehad lowautofluorescence,whichresultedinlowinterference.Inthe method developed here,good chromatographic resolution was achievedbetweentheanalyte(AlClPc)andtheI.S.(ZnPc).
Theprecisionofananalyticalprocedureexpressesaclose agree-mentbetweenaseriesofmeasurementsobtainedfrommultiple samplingofthesamehomogeneoussample,whereastheaccuracy ofananalyticalmethoddescribesaclosetestresultsobtainedbythe methodtothetrue(nominal)valueoftheanalyte.Forplasma sam-ples,intra-dayprecisionandaccuracyrangedfrom0.99%to5.50% and98.27%to107.31%,respectively.Forliversamples,intra-day precisionandaccuracyrangedfrom10.06%to12.71%and90.95% to103.15%,respectively.Inter-dayprecisionandaccuracyranged from6.24%to9.40%and93.81%to104.02%respectivelyforplasma samplesandrangedfrom11.44%to13.87%and99.48%to104.48%, respectively for theliversamples (Table 2).Theseresults were withinthelimitsestablishedbyFDAguidelinesforthevalidation ofbioanalyticalmethods[23].
Therecoveryvaluesobtainedforeachmatrixarepresentedin
Table3.Therecoverywasveryefficientforthestandardandthe I.S.inplasmaandliversamples.Themeanpercentagesrecovered were96.11%and99.27%forAlClPcand96.68%and95.76%forthe I.S.inplasmaandliversamples,respectively.Theextraction
proce-EU 0,00 20,00 Minutes 0,00 2,00 4,00 EU 0,00 20,00 Minutes 0,00 2,00 4,00 EU 0,00 5,00 10,00 Minutes 0,00 2,00 4,00 EU 0,00 50,00 100,00 Minutes 0,00 2,00 4,00 EU 0,00 50,00 100,00 Minutes 0,00 2,00 4,00 EU 0,00 50,00 100,00 Minutes 0,00 2,00 4,00 AlClPc AlClPc AlClPc AlClPc AlClPc AlClPc I.S. I.S. I.S. I.S.
A
B
C
E
F
D
Fig.2.Chromatogramsofsamplesof0.1mg/mlAlClPcloaded-PLANC(A)andAlClPcloaded-PLA-PEGNC(B).MiceplasmasamplesobtainedafterivadministrationofAlClPc loadedPLANC,5min(C)and12h(D).Miceliversamplesobtained20minafteradministrationof50l(E)and100l(F)of0.1mg/mlAlClPcPLANC.
Table2
Intra-andinter-dayprecisionandaccuracyofAlClPcdeterminedbybioanalyticalmethod.
Concentration Precision(RSD%)a Accuracy(%)a
Intra-day Inter-day Intra-day Inter-day
Plasma(ng/ml) 20 5.50 8.23 107.31 96.14 40 8.77 9.40 98.27 93.81 80 0.99 6.24 101.32 104.02 Liver(ng/g) 100 12.71 11.52 90.95 99.48 200 12.44 13.87 99.95 104.48 400 10.06 11.44 103.15 103.45
RSD,relativestandarddeviation.
an=6
durewasthesamefortheliverandplasmasamplesandexhibited highpercentagesof extractionof AlClPc fromthesamples.The extractionmethodologywassimple,fastandcost-effectivebecause onlyonesolventwasneededinlow volumesandthere wasno needforfurtherpurification.Furthermore,oneimportant advan-tageofthisbioanalyticaltechniquewasthatonly100lofplasma or200mgoftissueweresufficientforobtainingresultsinawide rangeofconcentrationsapplicabletobiodistribution,toxicology andpharmacokineticsstudies,whichwouldrequiresmall labora-toryanimalsorsamples,suchasbiopsies[14].TheZnPcwasfound tobeasuitableinternalstandardbecauseofitschemicalstability
[12,20],itssimilaritytoAlClPc’sstructureandisthefactthatitis easilyrecoveredfromplasmaandliversamples(>95%),asshown inTable3.
Table4listsdataforstabilitytests.Threesetsofplasma sam-plesatlow,mediumandhighconcentrationsofQCwereanalyzed inallstabilitytests,andtheresultsindicatedthattheAlClPcwas stable.Processedplasmaandliversampleswerestableforatleast 24hatroomtemperatureintheautosampler.Thesampleswere stableafterthethreefreeze–thawcycles.Theseresultssuggested thatplasmaandliversamplescouldbethawedandrefrozen with-outcompromisingtheintegrityandaccuracyofthesamples.The workingsolutions of AlClPcand I.S. were stablefor 30 days at −20◦C.The bench-topstability wasinvestigated toensurethat AlClPcremainedstableinplasmaandliversamplesatroom tem-peraturefor4h.TheAlClPcconcentrations(n=5)foundinplasma andtissuefrozenat−80◦Cwerenotstatisticallydifferent(p>0.05) fromtheconcentrationmeasuredatthebeginningofstudy.This ensembleofresultsconfirmedAlClPc’slong-termstabilityandits suitabilityasafluorescentdyeinNCformulationsandin biologi-calsamples.Theresultsobtainedonthestabilitystudiesindicated thatsamplescanbestoredat−80◦Cforthirtydayswithoutlossof theanalyte.ThefluorescenceresponseaftercontactofAlClPcwith
Table3
AbsoluterecoveryofAlClPcandZnPcfromplasmaandlivermatrices.
Concentrationa Recovery(%) AlClPc ZnPc(I.S.)b Plasma(ng/ml) 20 94.10 96.58 40 95.67 95.33 80 98.56 98.13 Mean 96.11 96.68 RSDc 1.92 1.18 Liver(ng/g) 100 96.32 89.96 200 101.53 96.34 400 99.96 100.97 Mean 99.27 95.76 RSDc 2.69 5.77 an=5. b ZnPcisinternalstandard. c Relativestandarddeviation.
solventwastime-independentbecauseofthesolventusedinthe method;incomparison,inapreviouslyreportedmethod,the con-tacttimebetweenthebiologicalsampleandsolventaffectedthe fluorescenceresponse[14].
Directchromatographicanalysisofthemajorityofthe phthalo-cyaninederivatives was difficultbecause of thepoor solubility ofthesecompoundsinthemobilephase, exceptforthemobile phases containing concentrated H2SO4 and dimethyl sulfoxide
[26].PhthalocyaninederivativeswereproblematicHPLCanalytes becauseof thepresenceof anextended regionthat is prone toadsorptionand-stackingphenomena.Theirchromatographic separationwaspossibleundertheconditionsofnormal-phaseand reversed-phaseHPLCbecauseofpolarandnonpolargroupsonthe molecule[25,26].Dimethylformamidewasmixedwithmethanol andacetonetoconstitutetheeluentphase.Itwassuccessfulin sol-ubilizingtheAlClPcandreducingtheadsorptiononthestationary phase,probablybyimprovinginteractionbetweentheanalyteand solvents.
3.1. Application
Nanocapsules with different superficial characteristics were preparedbythenanoprecipitationmethod[18,19]withmeansizes ofbetween155and256nm.TheAlClPccontentinPLA,PLA-PEGand PLA-CSNCwasdeterminedusingthevalidatedanalyticalmethod describedherein.
TheprecipitationofAlClPcwassometimesobservedafter prepa-ration of the NC; thus, the colloidal suspensions were filtered usinga0.8mmembranebeforetheAlClPcdeterminationineach nanocapsuleformulation.TheAlClPcpayloadinthenanocapsules rangedfrom35.3%to86.4%(Table5),asdeterminedpreviously forotherdrugs[19].ThehighertheAlClPcprecipitationinthe for-mulation,thelowertheefficiencyoftheencapsulationprocess.It wasinfluencedbytheNCcoatingcomposition.Chitosanreduces AlClPcassociationwithNC.However,drugloadingwasnot influ-encedbythecompositionoftheexternalsurfaceofnanocapsules at0.1mg/ml,indicatingthatAlClPcwasprobablyentrappedinthe oilycore.
Arecentlypublishedstudyreportedthevalidated spectropho-tometricandspectrofluorimetricmethodsforAlClPcquantification innanocarriers[17].However,themethoddescribedhereinalso allowedthequantificationoftheAlClPcwiththesamesensitivity andwithbetterselectivityinbiologicalsamples.Thebioanalytical method applied herein was able to determine AlClPc concen-trations in plasma and liver samples 20min after PLA NC was intravenouslyadministeredinmice.Abiodistributionstudywas usedtoinvestigatetheeffectofincreasingdosesofparticlesonthe miceplasmaandliversconcentrationsofAlClPc(Fig.3).Thedoses ofnanoparticlesadministeredtotheanimalswereparticularlyhigh tosaturateparticleuptakeby themononuclear phagocytic sys-tem(MPS).Itwasnecessarytodilutethesamplesofplasmafrom treatedmice5–10foldwithblankmiceplasmabeforethe
extrac-StabilitydataofAlClPcinplasmaandliversamples. Stability Nominal concentration Measured concentration (ng/ml) Precision (RSDa%) Accuracy(%) Plasma Benchtop4h 20ng/ml 20.95 7.05 104.27 40ng/ml 20.44 0.97 101.09 80ng/ml 75.51 2.42 94.38 Autosampler24h 20ng/ml 20.93 5.08 103.23 40ng/ml 43.22 7.46 108.05 80ng/ml 81.46 7.94 101.83
Freeze–thawthreecycles
20ng/ml 18.40 2.32 92.02 40ng/ml 38.36 2.36 95.89 80ng/ml 81.45 12.53 101.82 Liver Benchtop4h 100ng/g 101.15 1.55 101.15 200ng/g 198.76 6.30 99.38 400ng/g 402.64 8.25 100.66 Autosampler24h 100ng/g 100.75 8.10 100.75 200ng/g 209.05 9.48 104.52 400ng/g 428.8 9.91 107.20
Freeze–thawthreecycles
100ng/g 104.95 9.31 104.95
200ng/g 211.15 6.34 105.59
400ng/g 408.55 0.05 102.14
aRelativestandarddeviation.
tiontofittheAlClPcconcentrationinthebiologicalsampleswithin thecalibrationcurves.Thesametechniquewasusedfortheliver samples.Fig.3showsthatAlClPcconcentrationsintheplasmaand liversamplesareveryhighcomparedwiththeadministereddoses,
Fig.3.ConcentrationofAlClPcinmiceplasmaandliver20minafteradministration ofdifferentdosesofAlClPcinPLAformulation(n=4),*p<0.05.
0.1 0.2 0.4 0.8 1.6 AlClPc FREE AlClPc NC 48 42 36 30 24 18 12 6 3 0 Time (h) % of injected dose *** *** **
Fig.4.PlasmaAlClPcconcentration–timeprofilesafterintravenousadministration of150lof0.1mg/mlsolutionofAlClPcfreeand0.1mg/mlAlClPcinPLA-NC sus-pensioninmice(mean±SE,n=6).***p<0.001,**p<0.01,usingtwo-wayANOVA.
Table5
AlClPcassociationwithdifferentnanocapsuleformulationsdeterminedbythe ana-lyticalmethod. AlClPc nanocapsules (0.1mg/ml) Encapsulation efficiency(%) Drug loadinga(%) AlClPcb precipitated(%) PLA 71.9 100 9.2 PLA-PEG 86.4 100 7.1 PLA-CS 35.3 100 17.3
aDrugloadingwasdeterminedafterNCsuspensionfiltrationin0.8mfilter. bPrecipitaterecoveryafterfiltrationofNCsuspensionin0.8mfilter.
indicatingthatnanocapsulesconcentratethedyeintheplasmaand liver.Atthehigherdoses(30g/animal),asaturationofMPSseems tobereachedbecausetheAlClPcconcentrationinplasmaexhibited anexponentialincrease.TheplasmaandliverAlClPcconcentrations werefoundtobedose-dependent(Fig.3).
Thepharmacokineticprofilesobtainedafterintravenous admin-istrationofAlClPcsolutionandAlClPcloadedinPLA-NCareshown inFig.4.TheplasmaticconcentrationofAlClPcwassignificantly higher(p<0.001)thantheconcentrationsofthefreeAlClPcafter5, 20and60minofinjection.After3h,theplasmaticconcentration declinedsharplyuntil6h.After3hAlClPcconcentrationofboth formulationsweresimilarindicatingthattheAlClPcwasprobably releasedfromtheNC.Itcanbenotedthatnanocapsulesmodified theplasmaticprofileofAlClPc,suchasshowninTable6.TheAUC wasincreasedapproximately1.5timesandclearancewasreduced byencapsulatingthisdruginthesepolymericnanocarriers.
Table6
PharmacokineticparametersofAlClPcindifferentformulationsafterintravenous administration.
Parameter AlClPcfree AlClPcloaded inPLA-NC AUC0–48(ngmin/ml) 429.9*** 627.8
Clearance(ml/(minkg)) 1237.56*** 761.46
AUC,areaundercurve;NC,nanocapsules.Administereddoses:478g/kg free AlClPc,570g/kgAlClPcNC.
Inthepresentwork,onlyliverandplasmaassaysarereported; however,preliminaryresultsindicatedthatthismethodcouldalso beusedwithothertissues,suchasbrain,lungs,muscle,spleen, kidneysandtumortissueswiththesimilarLOQandrecovery(data notshown).
4. Conclusion
New, specific, accurate, and precise isocratic reverse-phase HPLCmethodswithfluorescencedetectionweredescribedforthe determinationofAlClPcindifferentpolymericnanocapsule formu-lationsandinbiologicalsamples.Themethodwasdevelopedto assessdataonthepre-clinicalevaluationofAlClPcasaPDT sen-sitizingagent.Themethodwassensitiveandrobustandcouldbe usedinfurtherbiodistributionandpharmacokineticstudiesand inroutineanalysisofPDTtherapywiththisphotosensitizer.Itis suggestedthatthismethodcouldbeusefulinAlClPcclinicaltrials.
Acknowledgements
TheauthorsthankUFOPforpersonalfinancialsupporttothe firstauthorandtheCNPq-BrazilresearchergrantprovidedtoVCF Mosqueira.ThisworkwasalsosupportedbyFAPEMIG (APQ-4403-07project)andbytheNANOBIOMGNetwork,MinasGerais,Brazil.
References
[1]Z.Z.Öztürk,N.kilinc¸,D.Atilla,A.G.Gürek,V.Ahsen,Recentstudiesin chem-icalsensorsbasedonphthalocyanines,J.Porphir.Phthalocyanines13(2009) 1179–1187.
[2] A.P.Castano, T.N.Deminova,M.R. Hamblin,Mechanisminphotodynamic therapy:partthree.Photosensitizerpharmacokinetics,biodistribution,tumor localizationandmodesoftumordestruction,Photodiag.Photodyn.Ther.2 (2005)91–106.
[3] M.Triesscheijn,P.Baas,J.H.Schellens,F.A.Stewart,Photodynamictherapyin oncology,Oncologist11(2006)1034–1044.
[4]M.M.Zuk,K.Tyczkowska,E.Ben-Hur,H.C.Newman,I.Rosenthal,S.W.Crane, Reversed-phaseliquidchromatographicdeterminationofchloroaluminum phthalocyaninetetrasulfonateincaninetissuesandfluids,J.Chromatogr.433 (1988)367–372.
[5]T.J.Dougherty,C.J.Gomer,B.W.Henderson,G.Jori,D.Kessel,M.Korbelik,J. Moan,Q.Peng,Photodynamictherapy,J.Natl.CancerInst.90(1998)889–905. [6] A.Juarrans,P.Jaén,F.Sanz-Rodriguez,J.Cuevas,S.González,Photodynamic therapyofcancer,basicprinciplesandapplications,Clin.Transl.Oncol.3(2008) 148–154.
[7]E.Aisling,O‘Connor,W.M.Gallaher,A.T.Byrne,Porphyrinandnonporphyrin photosensitizerinoncology:preclinicalandclinicaladvancesinphotodynamic therapy,Photochem.Photobiol.85(2009)1053–1074.
[8]Y. Sadzuk,F.Iwasaki,I.Sugiyamaa,K.Horiuchi,T.Hiranod,H.Ozawa, N. Kanayam,N.Okub,Phototoxicityofcoproporphyrinasanovelphotodynamic therapywasenhancedbyliposomalization,Toxicol.Lett.182(2008)110–114. [9]C.M.Cassidy,M.M.Tunney,P.A.McCarron,R.F.Donnelly,Drugdelivery strate-giesforphotodynamicantimicrobialchemotherapy:frombenchtoptoclinical practice,J.Photochem.Photobiol.B.95(2009)71–80.
[10]K.O‘Riordan,O.E.Akilov,T.Hassan,Thepotentialforphotodynamictherapy inthetreatmentoflocalizedinfections,Photodiag.Photodyn.Ther.2(2005) 246–262.
[11] P.Klusona,M.Drobeka,A.Kalaji,S.Zarubova,J.Krysa,J.Rakusan,Singlet oxy-genphotogenerationefficienciesofaseriesofphthalocyaninesinwell-defined spectralregions,J.Photochem.Photobiol.AChem.199(2008)267–273. [12]S.M.T.Nunes,F.S.Sguilla,A.C.Tedesco,Photophysicalstudiesofzinc
phthalo-cyanineandchloroaluminumphthalocyanineincorporatedintoliposomein thepresenceofadditives,Braz.J.Med.Biol.Res.37(2004)273–284. [13] M.Kyriazi,E.Alexandratou,D.Yova,M.Rallis,T.Trebst,Topical
photody-namictherapyofmurinenon-melanonaskincarcinomaswithchloroaluminum phthalocyanineandadiodolaser:pharmacokinetics,tumorresponseand cos-meticoutcomes,Photodermatol.Photoimmunol.Photomed.24(2008)87–94. [14] L.Lilge,C.O’Carrol,B.C.Wilson,Asolubilizationtechniqueforphotosensitizer quantificationinexvivotissuesamples,J.Photochem.Photobiol.B.39(1997) 229–235.
[15]D.K.Chatterjee,L.S.Fong,Y.Zhang,Nanoparticlesinphotodynamictherapy:an emergingparadigm,Adv.DrugDeliv.Rev.60(2008)1627–1637.
[16]D.Bechet,P.Couleaud,C.Frochot,M.-L.Viriot,F.Guillemin,M.Barberi-Heyob, Nanoparticlesasvehiclesfordeliveryofphotodynamictherapyagents,Trends Biotechnol.26(2008)612–621.
[17]M.P.Siqueira-Moura, F.L.Primo,A.P.F. Peti,A.C.Tedesco, Validated spec-trophotometric and espectrofluorimetric methods for determination of chloroaluminumphthalocyanineinnanocarriers,Pharmazie65(2010)9–14. [18]H.Fessi,F.Pusieux,J.P.Devissaguet,N.Ammoury,S.Benita,Nanocapsule
for-mationbyinterfacialpolymerdepositionfollowingsolventdisplacement,Int. J.Pharm.55(1989)R1–R4.
[19]V.C.F.Mosqueira,P.Legrand,G.Barrat,Surface-modifiedandconventional nanocapsulesasnovelformulationsforparenteraldeliveryofhalofantrine,J. Nanosci.Nanotechnol.6(2006)3193–3202.
[20]T.Bohn,T.Walczyk,Determinationofchlorophyllinplantsamplesbyliquid chromatographyusingzinc-phthalocyanineasaninternalstandard,J. Chro-matogr.A.1024(2004)123–128.
[21] ICHHarmonisedTripartiteguideline,InternationalConferenceon Harmonisa-tionofTechnicalRequirementsforRegistrationofPharmaceuticalsforHuman Use,ValidationofAnalyticalProcedures:TextandmethodologyQ29(R1),ICH SteeringCommittee,Geneva,Switzerland,2005,pp.1–13.
[22] CommitteeonCareandUseofLaboratoryAnimals,GuidefortheCareandUse ofLaboratoryAnimals,Natl.Inst.Health,Bethesda,DHHS,1985,Publ.No.(NIH), pp.86–123.
[23]USFoodandDrugAdministration,GuidanceforIndustry,BioanalyticalMethod Validation,CentreforDrugEvaluationandResearch(CDER),Rockville,2001. [24] C.S.Auletta,Acutesubchronicandchronictoxicology,in:M.J.Derelanko,M.A.
Hollinger(Eds.),CRCHandbookofToxicology,CRCPress,BocaRaton,1995,pp. 51–103.
[25] M.I.Uvarova,G.D.Brykina,O.A.Shpigun,Porphyrinsandphthalocyaninesin high-performanceliquidchromatography,J.Anal.Chem.55(2000)910–925. [26]M.Sommerauer,C.Rager,M.Hanack,Separationof
2(3),9(10),16(17),23(24)-tetrasubstitutedphthalocyanineswithnewlydevelopedHPLCphases,J.Am. Chem.Soc.118(1996)10085–10095.
