369

Farmaceutskahemijaianalitikalekova

PharmaceuticalChemistryandDrugAnalysis

370

SADRŽAJ–CONTENTS

FHA-P1

USAGEOFANTHOCYANESFORAUTHENTICATIONOFALIMENTARYPRODUCTSANDITSDRAWBACKS

- Eliza Łata, Agnieszka Fulczyk, Teresa Kowalska, Mieczysław Sajewicz 377

FHA-P2

RAČUNARSKIPROGRAMIZAREZOLUCIONIPARAMETARUMONOGRAFIJIEVROPSKEFARMAKOPEJEZAMETODUDERIVATIVNESPEKTROFOTOMETRIJE

COMPUTERPROGRAMSFORTHERESOLUTIONPARAMETERINTHEEUROPEANPHARMACOPEIAMONOGRAPHOFDERIVATIVESPECTROPHOTOMETRY

- Ana Homšek, Bojan Marković, Sote Vladimirov, Katarina Karljiković Rajić 378

FHA-P3

MEDICINSKAHEMIJAINHIBITORAHISTONDEACTILAZE6–INSILICOPRISTUPDIZAJNULEKOVA

MEDICINALCHEMISTRYOFHISTONEDEACETYLASE6INHIBITORS–INSILICODRUGDESIGNAPPROACHES

- Dušan Ružić, Katarina Nikolić, Danica Agbaba 380

FHA-P4

UTICAJOPERATIVNIHUSLOVAIMETODAEKSTRAKCIJENAANTIOKSIDANTIVNUAKTIVNOSTSMILJA(HELICHRYSUMITALICUM(ROTH)G.DONFIL.)

INFLUENCEOFOPERATIONALCONDITIONSANDEXTRACTIONMETHODSONANTIOXIDANTACTIVITYOFIMMORTELLEEXTRACTS(HELICHRYSUMITALICUM(ROTH)G.DONFIL.)

- Marina Kalić, Filip Balaž, Branislava Teofilović, Zorica Mrkonjić, Tijana Grebić, Aleksandra Jovanović Galović, Srđan Stojanović 382

371

FHA-P5

CHEMOMETRICEVALUATIONOFSOLVENTELUTIONSTRENGTHINREVERSED‐PHASETLCONRP2ANDRP8PLATESUSINGSELECTEDDRUGSANDMODELCOMPOUNDS

- Łukasz Komsta, Sebastian Gadowski, Joanna Wróbel, Karolina Skrzypiec 384

FHA-P6

PHOTOSTABILITYSTUDYOFAGOMELATINEBYUHPLC‐DAD/ESI‐Q‐TOF

- Jakub Trawiński, Robert Skibiński 385

FHA-P7

COLDLABELEDTRASTUZUMAB‐P‐SCN‐BN‐DTPAANDTRASTUZUMAB‐P‐SCN‐BN‐1B4M‐DTPACONJUGATES–PREPARATIONANDSPECTROSCOPICANALYSIS

- Marija Sterjova, Predrag Džodić, Petre Makreski, Emilija Janevik-Ivanovska 386

FHA-P8

PRIMENAPAMPATESTAUPROCENIPERMEABILNOSTIIRETENCIJEUKOŽIDERIVATAKORTIENSKEKISELINEMETILPREDNIZOLONAKAOPOTENCIJALNIHSOFTGLUKOKORTIKOIDA

THEUSEOFPAMPAFORSKINPERMEABILITYANDRETENTIONEVALUATIONOFMETILPREDNISOLONE–DERIVEDCORTIENICACIDDERIVATIVESASPOTENTIALSOFTGLUCOCORTICOIDS

- Jelena Bošković, Jelena Marković, Vladimir Dobričić, Olivera Čudina 387

FHA-P9

KOMPJUTERSKIDIZAJNAGONISTAIANTOGNISTA5‐HT2ARECEPTORA

COMBINEDLIGANDANDSTRUCTURE‐BASEDAPPROACHINSEARCHOF5‐HT2ARECEPTORAGONISTSANDANTAGONISTS

- Milica Radan, Mirjana Antonijević, Teodora Đikić, Katarina Nikolić, Danica Agbaba 389

372

FHA-P10

ISPITIVANJECITOTOKSIČNEAKTIVNOSTIAMINOKISELINSKIHESTARAVITAMINAENAĆELIJAMATUMORADOJKEIPLUĆA

CYTOTOXICACTIVITYOFAMINOACIDESTERSOFVITAMINEAGAINSTBREASTANDLUNGCANCERCELLLINES

- Žarko Gagić, Tatjana Srdić-Rajić, Katarina Nikolić, Danica Agbaba 391

FHA-P11

OPTIMIZACIJAHPLCMETODEZAODREĐIVANJEMETILPARABENAIPROPILPARABENAIZHIDROGELA

OPTIMIZATIONOFTHEHPLCMETHODFORTHEDETERMINATIONOFMETHYLPARABENANDPROPYLPARABENINHYDROGELS

- Miljan Jeremić, Slavica Sunarić 393

FHA-P12

RAZVOJHPLCMETODEZAODREĐIVANJESADRŽAJANEORGANSKIHNITRATAUIZOSORBID‐MONONITRATTABLETAMAPRIMENOMAQBDPRISTUPA

DEVELOPMENTOFHPLCMETHODFORTHEDETERMINATIONOFINORGANICNITRATEIMPURITYINISOSORBIDE‐MONONITRATETABLETSBYAQBDAPPROACH

- Nataša Milović, Ana Kalinić, Jasminka Ognjanović, Milena Rmandić, Anđelija Malenović 395

FHA-P13

MICELIZACIJABINARNIHSMEŠAŽUČNIHSOLINATRIJUM‐DEOKSIHOLATAINATRIJUM‐HIODEOKSIHOLATA

MICELLIZATIONOFTHEBINARYMIXTURESOFBILIARYSALTSSODIUM‐DEOXYCHOLATEANDSODIUM‐HYODEOXYCHOLATE

- Nemanja Todorović, Ivana Stojanović, Vesna Tepavčević, Mihalj Poša 397

373

FHA-P14

ISPITIVANJEUSLOVAZAENANTIOSEPARACIJUMOKSIFLOKSACINAIPOTENCIJALNEHIRALNENEČISTOĆE(R,R)‐IZOMERA

INVESTIGATIONOFCONDITIONSFORENANTIOSEPARATIONOFMOXIFLOXACINANDITSPOTENTIALCHIRALIMPURITY(R,R)‐ISOMER

- Marija Mitrović, Biljana Otašević, Ana Protić, Mira Zečević 399

FHA-P15

ISPITIVANJERETENCIONIHMEHANIZAMAAMLODIPINBESILATA,BISOPROLOLFUMARATAINJIHOVIHNEČISTOĆAUPOTREBOMTRIRAZLIČITEHILICKOLONE

INVESTIGATIONOFTHERETENTIONMECHANISMSOFAMLODIPINEBESYLATE,BISOPROLOLFUMARATEANDTHEIRIMPURITIESONTHREEDIFFERENTHILICCOLUMNS

- Irena Kasagić-Vujanović, Biljana Jančić-Stojanović, Darija Knežević, Darko Ivanović 401

FHA-P16

QbDPRISTUPURAZVOJUIVALIDACIJIHILICMETODEZAANALIZUAMITRIPTILINHIDROHLORIDAINJEGOVIHNEČISTOĆA

QbDORIENTEDDEVELOPMENTANDVALIDATIONOFTHEHILICMETHODFORTHEANALYSISOFAMITRIPTYLINEHYDROCHLORIDEANDITSIMPURITIES

- Irena Kasagić-Vujanović, Darija Knežević, Guro Forsdahl, Biljana Jančić-Stojanović 403

FHA-P17

ODREĐIVANJETELMISARTANAINJEGOVIHNEČISTOĆAUTABLETAMARP‐HPLCMETODOMSGRADIJENTNIMELUIRANJEM

DETERMINATIONOFTELMISARTANANDITSIMPURITESINTABLETSBYRP‐HPLCMETHODWITHGRADIENTELUTION

- Dragana Vukadinović, Vladimir Dobričić, Aleksandra Aranđelović, Danijela Radojičić, Jelena Maksić, Biljana Jančić-Stojanović, Olivera Čudina 405

374

FHA-P18

KARAKTERIZACIJABIOMOLEKULASAANTIBIOTIČKIMDEJSTVOMIZENDOFITAPHOMOPSISSPECIES

CHARACTERIZATIONOFBIOMOLECULESWITHANTIBIOTICACTIVITYFROMENDOPHYTEPHOMOPSISSPECIES

- Janko Ignjatović, Nevena Maljurić, Jelena Golubović, Miloš Petković, Matjaž Ravnikar, Borut Štrukelj, Biljana Otašević 407

FHA-P19

ADESIRABILITYBASEDMULTIOBJECTIVEAPPROACHFORMODELING,PREDICTINGANDOPTIMIZATIONOFEXPERIMENTALCONDITIONFORFORCEDDEGRADATIONOFROSUVASTATIN

- Maja Hadzieva Gigovska, Ana Petkovska, Jelena Acevska, Natalija Nakov, Blagica Manchevska, Packa Antovska, Sonja Ugarković, Aneta Dimitrovska 409

FHA-P20

DOEAPPROACHFOROPTIMIZATIONOFAGENERICHIGH‐PERFORMANCELIQUIDCHROMATOGRAPHYMETHODFORDETERMINATIONOFUNDECLAREDCOMMONCOUGHANDCOLDINGREDIENTSINNATURALPRODUCTS

- Marija Zafirova, Jelena Acevska, Liljana Ugrinova, Gabriela Petrovska-Dimitrievska, Vasil Karchev, Natalija Nakov, Katerina Brezovska, Aneta Dimitrovska, Suzana Trajkovic-Jolevska 410

FHA-P21

PRIMENAMETODAMULTIVARIJANTNIHANALIZAGLAVNIHKOMPONENTIIHIJERARHIJSKOGGRUPISANJAUISPITIVANJURAZDVAJANJAJEDINJENJAZIPRASIDONATEČNOMHROMATOGRAFIJOM

MODELINGOFLIQUIDCHROMATOGRAPHYSEPARATIONOFZIPRASIDONECOMPOUNDSUSINGMULTIVARIATEMETHODSOFPRINCIPALCOMPONENTANDHIERARCHICALCLUSTERINGANALYSIS

- Marija Čarapić, Katarina Nikolić, Adam Smolinski, Danica Agbaba 411

375

FHA-P22

ELEKTROHEMIJSKAKARAKTERIZACIJAREDOKSPROCESABRIMONIDINAIVARENIKLINA

ELECTROCHEMICALCHARACTERISATIONOFBRIMONIDINEANDVARENICLINEREDOXPROCESSES

- Valentina Radulović, Mara Aleksić, Vera Kapetanović, Danica Agbaba 413

FHA-P23

ODREĐIVANJESADRŽAJAEFEDRIN‐HIDROHLORIDAUFARMACEUTSKIMPREPARATIMAPRIMENOMRPHPLCMETODE

DEVELOPMENTANDVALIDATIONOFRPHPLCMETHODFORDETERMINATIONOFEPHEDRINEHYDROCHLORIDEINPHARMACEUTICALDOSAGEFORM

- Branka Ivković, Milkica Crevar Sakač, Jelena Purić, Milica Tasić 415

FHA-P24

ODREĐIVANJESADRŽAJAPARTENOLIDAUFARMACEUTSKIMPREPARATIMAPRIMENOMRPHPLCMETODE

DEVELOPMENTANDVALIDATIONOFRPHPLCMETHODFORDETERMINATIONOFPARTENOLIDEINPHARMACEUTICALDOSAGEFORM

- Branka Ivković, Milkica Crevar Sakač, Biljana Fidanovski, Dragana Stanković 417

FHA-P25

EQUIVALENCETESTSASANEWSTATISTICALAPPROACHINANALYTICALMETHODTRANSFERS

- Aleksandra Petrovska, Marija Velickovska, Natasa Anevska Stojanovska, Sonja Ugarković 419

376

FHA-P26

ODREĐIVANJESADRŽAJAKOFEINAUKOZMETIČKIMPREPARATIMAZAKOSUPRIMENOMEKSTRAKCIJENAČVRSTOJFAZIIHPLCMETODE

ASSESSMENTOFCAFFEINECONTENTINHAIRCAREPRODUCTSBYSOLIDPHASEEXTRACTIONANDHPLCMETHOD

- Kristina Mladenov, Slavica Sunarić 420

FHA-P27

VALIDACIJAHPLC‐UVMETODEZAODREĐIVANJEMOKSIFLOKSACINAICIPROFLOKSACINAUPERITONEALNOJTEČNOSTIKODPACIJENATANAPERITONEALNOJDIJALIZI

VALIDATIONOFHPLC‐UVMETHODFORDETERMINATIONOFMOXIFLOXACINANDCIPROFLOXACININPERITONEALFLUIDOFPATIENTSONPERITONEALDIALYSIS

- Maja Koraćević, Predrag Džodić, Radmila Veličković Radovanović, Tatjana Cvetković 422

FHA-P28

UTICAJMICELARAZLIČITOGNAELEKTRISANJAKAOSIMULIRAJUĆIHSISTEMABIOMEMBRANANAJONIZACIJURUPATADINA

THEEFFECTSOFDIFFERENTLYCHARGEDMICELLESASBIOMEMBRANEMIMETICSYSTEMSONTHEIONIZATIONOFRUPATADINE

- Marija Popović Nikolić, Gordana Popović, Kristina Stojilković, Maja Dobrosavljević, Danica Agbaba 424

377

Arh.farm 2018;68: 377 FHA-P1

USAGEOFANTHOCYANESFORAUTHENTICATIONOFALIMENTARY

PRODUCTSANDITSDRAWBACKS

Eliza Łata, Agnieszka Fulczyk, Teresa Kowalska, Mieczysław Sajewicz

Institute of Chemistry, University of Silesia, Katowice (Poland)

Authentication of alimentary products based on botanical raw materials (e.g.,

fruits, grains, vegetables etc.) is often based on identification and quantification ofanthocyanes contained therein. For authentication purpose,we need fast proceduresandoneofthemisscreeningoftheseproductswithuseofthin‐layerchromatography(TLC).

Itwas the aim of this study to show vulnerability of anthocyanes analyzed bymeansofTLCagainststationaryphasesofdifferentactivity(cellulosepowder,RP‐C18)andmixedmobilephasesemployingorganicacids(formicacid,aceticacid)andhence,alimitedperformancethereofasauthenticationmarkers.Tothiseffect,weusedcyaninand keracyanin (two anthocyanines), andpelargonidin and delphinidin (twoanthocyanidines)asfourphytochemicalstandards.Partialhydrolyticdecompositionofanthocyanines was demonstrated by the results originating from TLC, massspectrometry with use of the TLC‐MS interface and from using the sugars‐specificvisualizationreagentPABA.Moreover,calibrationcurveswereelaboratedforthefourtest anthocyanes and then these compounds were identified and quantified in theselected alimentary samples (fruit juices and herbal infusions). It was demonstratedthat authentication of alimentary products with use of anthocyanes as authenticitymarkerscanresult inasemi‐quantitativeresponseonlyandinpractical impossibilitytoconfirmwithfullconfidencethepresenceofaparticularanthocyaneinascrutinizedsample of a fruit juice or herbal infusion. In conclusion, there is a warning thatauthentication of alimentary products containing anthocyanes can occasionally bemisleading.

378

Arh.farm 2018;68: 378-379 FHA-P2

RAČUNARSKIPROGRAMIZAREZOLUCIONIPARAMETARUMONOGRAFIJIEVROPSKEFARMAKOPEJEZAMETODU

DERIVATIVNESPEKTROFOTOMETRIJE

AnaHomšek1,BojanMarković2,SoteVladimirov2,KatarinaKarljikovićRajić1

1Katedrazaanalitičkuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet,

2Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet(Srbija)

Evropska farmakopeja (EP 9) propisuje monografiju za metodu derivativne

spektrofotometrije koju kаrаktеrišе difеrеnciјаciја оsnоvnih аpsоrpciоnih spеktаrа udеrivаtivne spеktre оdgоvаrајućеg izvоdа i od posebnog značaja je za primene ufarmaceutskim analizama. U monografiji je propisan rezolucioni parametar kojipredstavlja odnos amplituda (A/B) iz derivativnog spektra drugog izvoda za modelsistem toluen/metanol. Rеzоluciоni pаrаmеtаr izrаčunаt u sоftvrеskоm prоgrаmuCintral jeupoređensаvrеdnоstimаdоbiјеnimuprоgrаmuOriginPro8iuprethodnimistraživanjimauprogramuSpectralVer.1.70.

PrоpisаnmоdеlrаstvоrprеmаEPmоnоgrаfiјi јеtоluеnumеtаnоlu0,02%V/V.Snimlјеniоsnоvnispеktrizapetrastvoraprevedenisuuderivativnedrugog izvodauprоgrаmima Cintral i OriginPro8. Nakon toga birani su odgovarajući parametri zakorekciju šuma u oba programa (opisni u Cintral‐u: najmanji, srednji i najveći;numeričkiuOriginPro8:5,7i9).

DirеktnоizrаčunаtdrugiizvоddоbiјеnuCintral‐udavaojesrеdnjuvrednostА/B0,6548uznајvеćuvrеdnоstRSD.Nајmаnjоmisrеdnjоmkоrеkciјоmšumаpоvеćаvаlasе srеdnjа vrednost А/B uz smаnjеnjе RSD, dоk je nајvеćоm korekcijom dobijenasrеdnjа vrednost А/B bila znаčајnо smаnjena. Dirеktnо izrаčunаt drugi izvоd uOriginPro8dаvaojenеštоnižusrеdnjuvrednostА/B(0,6408)оdCintral‐а iznаčајnоmаnjuRSD.Kоrеkciја šumа sа 5 nije imala uticаја nа srеdnju vrednostА/B, sаmо jesmаnjivala RSD, dоk je 7 smаnjivala srеdnju vrednost А/B i davala sličnu RSD, аvrеdnоsti9ivеćе,zakorekcijušuma,nisubileprimеnlјivе.

PrоgrаmiCintraliOriginPro8davalisusličnеvrеdnоstizаrеzоluciоnipаrаmеtаrА/BpričеmuјеzаdоvоlјеnprоpisEP(А/Bniјеmаnjiоd0,2).Srеdnjаkоrеkciјаšumа(Cintral)bilajeupоrеdivаsа7kоrеkciјоm(OriginPro8)iоbеsuоptimаlnеzаdоbiјаnjеdigitаlnih dеrivаtivnih spеktаrа. Dobijeni rezultati značajni su za procenumogućnostipoređenja kod međulaboratorijskih analiza i transfera metoda zaodređivanjaprimenomderivativnespektrofotometrije.

379

COMPUTERPROGRAMSFORTHERESOLUTIONPARAMETERINTHE

EUROPEANPHARMACOPEIAMONOGRAPHOFDERIVATIVESPECTROPHOTOMETRY

AnaHomšek1,BojanMarković2,SoteVladimirov2,

KatarinaKarljiković Rajić1

1DepartmentofAnalyticalChemistry,UniversityofBelgrade‐FacultyofPharmacy,2DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐

FacultyofPharmacy(Serbia) European Pharmacopeia (EP) prescribes the monograph for derivative

spectrophotometry which is characterized by the differentiation of zero‐orderabsorption spectra intoderivative spectraof correspondingorderand is thereforeofgreat significance for application in pharmaceutical analysis. In the monographresolution parameter, which represents the ratio of amplitudes (A/B) of the secondorder for the model solution toluene/methanol, is prescribed. The resolutionparameter calculated in software program Cintral was compared to the valuescalculated in OriginPro8 and to the ones obtained from the previous research inSpectralVer.1.70.

The prescribed model solution according to EP monograph is toluene inmethanol 0.02% V/V. Zero‐order spectra for the five solutions were recorded andtransformedintosecondorderderivativespectrainbothCintralandOriginPro8.Thenthe smoothing parameters were chosen (descriptive in Cintral: light, medium andheavy;numericinOriginPro8:5,7and9).

SecondorderspectradirectlycalculatedbyCintralgavemeanvalueA/B0.6548withthehighestRSD.LightandmediumsmoothingincreasedthemeanvalueA/BwithadecreaseofRSD,whereasheavysmoothingsignificantlydecreasedmeanvalueA/B.Directly calculated second order in OriginPro8 gave slightly lower mean value A/B(0.6408)thanCintralandsignificantlylowerRSD.Smoothing5didn'timpactthemeanvalueA/B,butdecreasedRSD,whereas7decreasedmeanvalueA/BandgavesimilarRSD,butsmoothings9andhigherwerenotapplicable.

Programs Cintral andOriginPro8 gave similar values for resolution parameterA/B,whichisasprescribedinEP(A/Bnotlessthan0.2).Mediumsmoothing(Cintral)was comparable to smoothing 7 (OriginPro8) and both are optimal to obtain digitalderivative spectra. Obtained results were significant for the assessment of thecomparison possibility in inter‐laboratory analysis and method transfer for assayanalysisusingderivativespectrophotometry.

380

Arh.farm 2018;68: 380-381 FHA-P3

MEDICINSKAHEMIJAINHIBITORAHISTONDEACTILAZE6–IN

SILICOPRISTUPDIZAJNULEKOVA

DušanRužić,KatarinaNikolić,DanicaAgbaba

Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet(Srbija)

Hemija postranslacionihmodifikacija histona, kao i njihov uticaj na ekspresiju

gena predstavlja jedan od najizazovnijih procesa koji se izučava u kancerskojepigenetici. Kovalentne modifikacije, kao što su acetilovanje i deacetilovanje histonamenjajuarhitekturuhromatinaimogudovestidorazličitihćelijskihodgovora.Od2006.godine,registrovanoje5inhibitorahistondeacetilaza,efikasnihuterapijihematološkihmaligniteta. Medicinski hemičari posvećuju posebnu pažnju selektivnostinovodizajniranih jedinjenja ka HDAC6 izoformi, koja je jedinstveno lokalizovana ucitoplazmiiutičenadinamskeprocesecitoskeleta.

Metode dizajna lekova bazirane na hemijskim strukturama poznatih HDAC1iHDAC6 inhibitora (pristup zasnovan na strukturi liganada) unapredile su našerazumevanjestrukturnihkarakteristikaneophodnihzapotentnuHDAC6inhibiciju.Dabi se kvantifikovao odnos između strukture i potentnosti različitih HDAC inhibitora,primenili smo 3D‐QSAR studiju (trodimenzionalnu studiju odnosa strukture iaktivnosti) sa već publikovanim strukturama HDAC1 i HDAC6 inhibitora. Hemijskastruktura skriptaida i njegovi izračunati tridimenzionalni deskriptori su korišćeni zapretragu novih fragmenata sa sličnim hemijskim osobinama kao inaftalimidno jezgo(dizajn zasnovan na strukturi fragmenta). Predviđene HDAC1 i HDAC6 aktivnostinovodizajniranih jedinjenja su dobijene korišćenjem validiranih 3D‐QSARmodela. Zadalje studije, odabrana su ona jedinjenja sa poboljšanom in silico selektivnošćuusmerenoj ka HDAC6 izoformi. Način vezivanja novih jedinjenja je ispitan studijamamolekulskogdokingaiupoređensanačinomvezivanjapoznatihHDACinhibitora.

Kombinovani pristupi zasnovani na strukturi liganda, fragmenata i strukturivezivnogmestasuuspešnoprimenjeniunašojgrupizapronalaženjenovihihemijskihatraktivnihHDAC6inhibitora.Uočenojedasupredviđeneaktivnostipomoću3D‐QSARstudije za najbolje rangirana dizajnirana jedinjenja u korelaciji sa rezultatima studijevirtuelnog dokinga. Ovakav kombinovani protokol povećava šansu pronalaženja HITjedinjenja kao selektivnog HDAC6inhibitora, što će u narednim istraživanjima bitipotvrđenoinvitrostudijamanovosintetisanihjedinjenjaunašojlaboratoriji.

381

MEDICINALCHEMISTRYOFHISTONEDEACETYLASE6INHIBITORS–

INSILICODRUGDESIGNAPPROACHES

DušanRužić,KatarinaNikolić,DanicaAgbaba

DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐FacultyofPharmacy(Serbia)

Thechemistryofhistoneposttranslationalmodificationsandtheir influenceon

gene expression present one of the most challenging processes studied in cancerepigenetics.Thecovalentmodifications,suchashistoneacetylationanddeacetylationalter the chromatin architecture and lead to different cellular responses. Since2006,therehavebeenfivehistonedeacetylase(HDAC)inhibitorsclinicallyapprovedforthehaematologicalcancers.Medicinalchemistspayparticularattentiontotheselectivityofnewly designed compounds against HDAC6 isoform, as it is uniquely located in thecytoplasmandcontrolsthedynamicsofthecytoskeleton.

Drugdesignmethodologiesbasedon theknownHDAC1andHDAC6 inhibitorsstructures (ligand‐based approach) improved our understanding of the structuralrequirements needed for potentHDAC6 inhibitors. To quantify the relation betweenthe structure and potency in a group of diverseHDAC inhibitors,we performed 3D‐QSAR(QuantitativeStructure‐ActivityRelationship)studieswithpublishedHDAC1andHDAC6 inhibitors. The structure of scriptaid and its derived three‐dimensionaldescriptors were used for searching of novel fragments with the similar chemicalpropertiesasitsnaphthalimidecore(fragment‐baseddesign).

ThepredictedHDAC1andHDAC6activitiesofnewlydesignedcompoundswereobtained by validated 3D‐QSAR models. We selected only those compounds withimproved in silico selectivity towardHDAC6 isoform for further studies.Thebindingmodes of novel compounds were introspected by molecular docking studies andcomparedwiththeknownHDACinhibitorsbindingmodes.

Thecombinedligand‐based,fragment‐basedandstructure‐basedmethodologieswere successively applied in our group to discover novel and chemically attractiveHDAC6inhibitors.Weobservedthatthepredictedpotencyofthetop‐rankeddesignedHDAC6 inhibitorsby3D‐QSARstudiesarecorrelatedwith thevirtualdockingresults.The combined protocol increases the hit rate in the discovery of selective HDAC6inhibitors, which will be further examined by in vitro studies of synthesizedcompoundsinourlaboratory.

382

Arh.farm 2018;68: 382-383 FHA-P4 UTICAJOPERATIVNIHUSLOVAIMETODAEKSTRAKCIJENA

ANTIOKSIDANTIVNUAKTIVNOSTSMILJA(HELICHRYSUMITALICUM(ROTH)G.DONFIL.)

MarinaKalić1,FilipBalaž1,BranislavaTeofilović1,ZoricaMrkonjić1,TijanaGrebić2,AleksandraJovanovićGalović1,SrđanStojanović3

1UniverzitetPrivrednaakademijauNovomSadu‐FarmaceutskifakultetNoviSad(Srbija),2LjekarnaJoukhadar(Hrvatska),3Internacionalnicentarza

profesionalnestudije(Srbija)Smilje (Helichrysum italicum (Roth) G. Don fil.) je rasprostranjeno u

mediteranskompodručju.Smilje, sadržiaktivneprincipe isekundarnemetabolitekaošto su fenolne kiseline, flavonoidi, pironi, triterpenoidi, seskviterpeni, acetofenoni,floroglucinoli i male količine etarskog ulja. Cilj ovog rada bio je da se testiraantioksidativnaaktivnostekstrakatasmiljadobijenihodrazličitihpolarnihinepolarnihrastvarača.

Ekstrakcije su izvedene vodom, metanolom, etil acetatom, hloroformom,acetonom,n‐heksanomimetilhloridomtokomrazličitihvremenskihinterval(10,30i60 minuta), a stepen usitnjenosti bio je 0,3 i 2 mm. Antioksidativna aktivnost jetestirana spektrofotometrijskom metodom koja meri ukupan sadržaj fenola iflavonoida,kaoiinhibitorneaktivnostiDPPH(2,2–difenil–1–pikrilhidrazil)radikala.

Rezultati supokazali da suanalizirani ekstrakti imali značajnuantioksidativnuaktivnost.Ukupansadržajfenolakrećeseod21,26do53,61mgGAE/gSE.SviekstraktisupokazalidobruantioksidativnuaktivnostsaIC50vrednošćuurasponuod21,38do71,18 μg/mL. Sa druge strane, svi ekstrakti su pokazali generalno nizak sadržajukupnihflavonoida.

Utvrđeno je da povećano vreme ekstrakcije, polariteta rastvarača i stepenusitnjenosti povećavaju kvalitet ekstrakta u smislu sadržaja fenolnih komponenti iantioksidantivnihefekata.

383

INFLUENCEOFOPERATIONALCONDITIONSANDEXTRACTIONMETHODSONANTIOXIDANTACTIVITYOFIMMORTELLEEXTRACTS

(HELICHRYSUMITALICUM(ROTH)G.DONFIL.)

MarinaKalić1,FilipBalaž1,BranislavaTeofilović1,ZoricaMrkonjić1,TijanaGrebić2,AleksandraJovanovićGalović1,SrđanStojanović3

1UniversityBusinessAcademyinNoviSad‐FacultyofPharmacyNoviSad

(Serbia),2PharmacyJoukhadar(Croatia),3InternationalCenterforProfessionalStudies(Serbia)

Immortelle(Helichrysumitalicum,(Roth)G.Donfil.)iswidelydistributedinthe

Mediterranean region. Immortelle contains important metabolites such as phenolicacids, flavonoids, pyrones, triterpenoids, sesquiterpenes, acetophenones,phloroglucinols,andsmallamountofessentialoil.Theaimofthisworkwastotesttheantioxidantactivityofimmortelleextractsobtainedfromdifferentpolarandnon‐polarsolvents.

Theextractionwasperformedwithwater,methanol,ethyl‐acetate,chloroform,acetone,n‐hexaneandmethylenechlorideduringdifferentperiodsoftime(10,30and60minutes)and thedegreeof fragmentationwas0.3and2mm.Antioxidantactivitywas tested by spectrophotometric method measuring total phenolic and flavonoidcontent and inhibitory activity of DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) radical. Theresultsshowedthatanalyzedextractshadsignificantantioxidantactivity.Totalphenolcontentrangedfrom21.26–53.61mgGAE/gDE.AllextractsshowedgoodantioxidantactivitywithanIC50valueintherangefrom21.38to71.18μg/ml.Ontheotherhand,allextractsshowedgenerallylowcontentoftotalflavonoids.

It was found that increased time of extraction, solvent polarity and degree ofcomminutionofthedrugincreasethequalityoftheextractsintermsofthecontentofphenoliccomponentsandantioxidanteffects.

384

Arh.farm 2018;68: 384 FHA-P5

CHEMOMETRICEVALUATIONOFSOLVENTELUTIONSTRENGTHINREVERSED‐PHASETLCONRP2ANDRP8PLATESUSINGSELECTED

DRUGSANDMODELCOMPOUNDS

ŁukaszKomsta,SebastianGadowski,JoannaWróbel,KarolinaSkrzypiec

DepartmentofMedicinalChemistry,MedicalUniversityofLublin(Poland)

To investigate in experimental conditions the eluotropic strength on RP2 and

RP8 plates for 7 chromatographic grade solvents (acetone, acetonitrile, dioxane,ethanol, isopropanol,methanol,tetrahydrofuran),aswellasforwateritself.35modelcompounds(includingdrugs)wereusedasatestsetintheinvestigation.Theuseofamodern chemometricmixturedesign conceptallowed to estimate each solvent effectseparatelyfromtheotherresultswithasmallerror(about0.05ofRMvalue).Principalcomponent analysis revealed that only one parameter (mean eluotropic strength)explainsoverallvariabilityintheretentiondatasets.Thebootstrappingwasalsousedto check thedistributionanduncertaintyof theobtainedvalues. It canbe concludedthatallsolventshavequitesimilarelutionstrengthforbothadsorbents.TheobtainedvaluescanbeusedasareferenceduringoptimizationofTLCsystems.

385

Arh.farm 2018;68: 385 FHA-P6

PHOTOSTABILITYSTUDYOFAGOMELATINEBYUHPLC‐DAD/ESI‐Q‐TOF

JakubTrawiński,RobertSkibiński

DepartmentofMedicinalChemistry,MedicalUniversityofLublin(Poland)

According to the European Pharmacopoeia, over 250 active pharmaceuticalingredients are classified as photolabile. This fact involves two major risks. Firstly,exposure of the photolabile pharmaceuticals to the solar or artificial radiation couldlead to lossof theirpharmacologicalactivity.Secondly, such interactioncould lead toformation of transformation products (TPs), possibly more toxic than the parentmolecule. The aim of this study was an investigation of the direct photolysis ofagomelatine under the ICH‐recommended conditions. Agomelatine, a novel atypicalantidepressant, is a potent agonist of melatonin receptors, and 5‐HT2C receptorantagonist.

Aqueoussolutionofagomelatinewasirradiated(0–150min)underthexenonlamp, equipped with D65 filter. The applied conditions imitated full UV‐Vis solarspectrum. Obtained sampleswere then analyzedwith the use of UHPLC‐DAD/ESI‐Q‐TOFcoupledsystem.RP‐18columnandgradientelutionofmobilephaseconsistingofwaterwithadditionof0.1%formicacidandacetonitrilewereused.ACD/LabsPerceptasoftwarewasusedtocalculateacutetoxicitytorodentsoftheidentifiedcompounds.

During the experiment over half of the initial agomelatine concentration wasdecomposed.Reactionkineticsfittedthefirst‐ordermodel,andthedegradationhalf‐lifewas 131.69 min. Six main TPs were found, and their chemical structures wereelucidated. All of the identified TPs were found as a agomelatine photooxidation ordemethylationproducts.Additionally, the insilicoevaluationof theiracutetoxicitytorodents was performed. The results, obtained with the use of six models weresubsequently submitted to PCA, in order to visualize relationships between the TPsproperties.

Results of this study demonstrated that agomelatine is not a photostablemolecule, and pharmaceutical formulations containing this compound should beprotected from light.Majorityof the identifiedTPswere formedasa consequenceofagomelatinehydroxylationoroxidation.PCA facilitated comparisonof toxicityofTPsandtheparentcompound.

386

Arh.farm 2018;68: 386 FHA-P7 COLDLABELEDTRASTUZUMAB‐P‐SCN‐BN‐DTPAANDTRASTUZUMAB‐P‐SCN‐BN‐1B4M‐DTPACONJUGATES–

PREPARATIONANDSPECTROSCOPICANALYSIS

MarijaSterjova1,PredragDžodić2,PetreMakreski3,EmilijaJanevik‐Ivanovska1

1University‘GoceDelčev’‐FacultyofMedicalSciences,2DepartmentofPharmacy,UniversityofNiš‐FacultyofMedicine(Serbia),3Instituteof

Chemistry,Ss.CyrilandMethodiusUniversity‐FacultyofNaturalSciencesandMathematics

The importance of immunoconjugates in treatment of various cancers was

motivationforusto formulateastablecold labeledtrastuzumabconjugateswith twobifunctional chelators (BFCAs) (p‐SCN‐Bn‐1B4M‐DTPA(2‐(4‐isothiocyanatobenzyl)‐6‐methyl‐diethylene‐triaminepentaacetic acid and p‐SCN‐Bn‐DTPA(2‐(4‐izothiocyanatobenzyl)‐diethylenetriaminepentaacetic acid)). The labeling with non‐radioactive LuCl3 and YCl3 is important to determine the possible physicochemicalchangesinthestructureofimmunoconjugatesaftermetalbinding.ATR‐IR(Attenuatedtotal reflectance‐infrared) and Raman spectroscopy as powerful and non‐destructivetechniquesareappropriateforverificationofpossiblesecondarystructurechancesoftrastuzumabafterconjugationandlabeling.

Anti‐HER2/neumonoclonal antibody trastuzumabwas conjugatedwithp‐SCN‐Bn‐DTPA,p‐SCN‐Bn‐1B4M‐DTPAinratioof1:10and1:50andlyophilizedtosolidstate.The freeze dried conjugates were labeled with cold LuCl3 and YCl3. The retainedsecondarystructureoftheantibodywasprovenbyspectroscopicanalysiswithATR‐IRand Raman spectroscopy and comparedwith purified trastuzumab from commercialproductHerceptin®.TheATR‐IRandRamanspectraof four sampleshave shown thepresence of characteristic amide bands and retained native IgG1 structure of theantibodyprincipallycomposedofβ‐sheets.CharacteristicamideIbandat~1670cm‐1andamideIIIband(1230‐1300cm‐1)weredetectedinRamanspectra.IRspectraalsocontaintheamideI(1700‐1600cm‐1),amideII(1480‐1575cm‐1)andamideIIIbands(1255‐1244cm‐1)specificforsecondarystructureoftheproteins.

Nosignificantchanges inantibodystructureaftercold labelinggivesusahopefor further radiolabeling of immunoconjugates with177LuCl3 and 90YCl3anddevelopmentofradioimmunotherapeuticsanddiagnosticproductsactiveagainstHER2positivebreasttumors.

387

Arh.farm 2018;68: 387-388 FHA-P8

PRIMENAPAMPATESTAUPROCENIPERMEABILNOSTIIRETENCIJEUKOŽIDERIVATAKORTIENSKEKISELINEMETILPREDNIZOLONA

KAOPOTENCIJALNIHSOFTGLUKOKORTIKOIDA

JelenaBošković,JelenaMarković,VladimirDobričić,OliveraČudina

Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu–Farmaceutskifakultet(Srbija)

Amidi ili estri kortienske kiselinemetilprednizolona predstavljaju potencijalne

soft glukokortikoide sa manje izraženim neželjenim efektima u odnosu nakonvencionalne glukokortikoide. Retencija i permeabilnost su važne osobine softglukokortikoidazaprimenunakožukojemoguznačajnouticatinanjihovuaktivnostipojavuneželjenihefekata.Jednaodinvitrometodakojasenajčešćekoristizaprocenuovih osobina je PAMPA (Parallel Artificial Membrane Permeability Assay). Cilj ovogradajeprocenapermeabilnostiiretencijeukožipetamida(MPMA,MPMAB,MPMAIB,MPMGB i MPPA) i jednog tioestra (MPEMP) kortienske kiseline metilprednizolonaprimenomPAMPAtesta.

Procena permeabilnosti i retencije u koži izvršena je na hidrofobnojMilliporePVDF PAMPA mikrotitarskoj ploči sa 96 odeljaka. Praćena je difuzija ispitivanihjedinjenja kroz membranu koju čine 70 % silikonsko ulje i 30 % izopropilmiristat.Koncentracijeupolaznimrastvorima,donorskimiakceptorskimodeljcimaodređenesuprimenomLC‐MS/MSmetode.

Primenom PAMPA testa određeni su koeficijenti permeabilnosti (logPe) iretencije (R). Vrednosti logPe su u opsegu od ‐6,81 do ‐5,09, dok su vrednosti R uopsegu 1,52 ‐ 65,25. Jedinjenje sa najnižom vrednošću logPe je MPMGB, dok su zaMPEMPodređenenajviševrednostiparametaralogPeiR,teseodovogderivatamoguočekivatinajvećapermeabilnostiretencijaukoži.

Permeabilnostiretencijaukožipetamida(MPMA,MPMAB,MPMAIB,MPMGBiMPPA)ijednogtioestra(MPEMP)kortienskekiselinemetilprednizolonaprocenjenisuprimenomPAMPAtesta.NajviševrednostiparametaralogPeiRsudobijenezaMPEMP,teseovajderivatizdvajakaonajpovoljnijizaprimenunakožu.

388

THEUSEOFPAMPAFORSKINPERMEABILITYANDRETENTIONEVALUATIONOFMETILPREDNISOLONE–DERIVEDCORTIENICACID

DERIVATIVESASPOTENTIALSOFTGLUCOCORTICOIDS

JelenaBošković,JelenaMarković,VladimirDobričić,OliveraČudina

DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐FacultyofPharmacy(Serbia)

Amidesorestersofmethylprednisolone‐derivedcortienicacidarepotentialsoft

glucocorticoids with fewer side effects in comparison to traditional glucocorticoids.Retention and permeability are important properties of soft glucocorticoids for localskinapplicationwhichcouldsignificantlyinfluencetheiractivityandoccurrenceofsideeffects.PAMPA(ParallelArtificialMembranePermeabilityAssay) isoneof themostlyusedinvitromethodsfortheestimationoftheseproperties.Theaimofthisstudywasestimationofskinpermeabilityandretentionoffiveamides(MPMA,MPMAB,MPMAIB,MPMGB and MPPA) and one thioester (MPEMP) of metilprednisolone ‐ derivedcortienicacidusingPAMPA.

Estimation of skin permeability and retentionwas performed on hydrophobicMilliporePVDFPAMPAmicrotiter96‐wellplate.Diffusionoftestedcompoundsthroughmembrane, consisting of 70% silicon oil and 30% isopropyl myristate, was tested.Concentrations in starting solutions, aswell as in donor and acceptor compartmentsweredeterminedusingLC‐MS/MSmethod.

PAMPA permeability coefficients (logPe) and retention (R) were determined.logPevaluesrangedfrom‐6.81to‐5.09,whereasRvaluesrangedfrom1.52to65.25.Derivativewith the lowestvalueof logPewasMPMGB,whereas thehighestvaluesoflogPe and R were determined for MPEMP. Therefore, highest skin permeability andretentioncouldbeexpectedfromMPEMP.

Skin permeability and retention of five amides (MPMA, MPMAB, MPMAIB,MPMGB and MPPA) and one thioester (MPEMP) of metilprednisolone ‐ derivedcortienicacidwasestimatedbyuseofPAMPA.ThehighestvaluesoflogPeandRwerecalculated for MPEMP, which could be considered the best candidate for skinapplication.

389

Arh.farm 2018;68: 389-390 FHA-P9 KOMPJUTERSKIDIZAJNAGONISTAIANTOGNISTA5‐HT2A

RECEPTORA

MilicaRadan,MirjanaAntonijević,TeodoraĐikić,KatarinaNikolić,DanicaAgbaba

Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu‐Farmaceutski

fakultet(Srbija)

Serotoninski 5‐HT2A receptori su uključeni u mnogobrojne fiziološke i

patofiziološke procese. Strukturno različiti ligandi (agonisti, antagonisti i inverzniagonisti) dovode do različitih konformacionih promena ovih receptora, izazivajućibrojnebiološkeodgovore.

Da bi se molekul ponašao kao agonista/antagonista potrebno je da posedujerazličite funkcionalne grupe i specifične interakcije sa određenim aminokiselinama uvezujućem mestu receptora. Razumevanje i objašnjavanje različitosti u strukturi ivezivanju za receptor, kod agonista i antagonista,može biti od značaja za racionalnidizajnnovihlekova.

Za razumevanje strukturnih razlikau farmakoforama, kao i kinetici vezivanja izmeđuagonista iantagonistakorišćeni su ligand‐based i structure‐basedpristupi.3D‐QSAR(3D‐quantitative structureactivityrelationship)studije su izvođenenagrupamaod 79 agonista i 90 antagonista. Uporedo su odrađene četiri simulacijemolekularnedinamike: serotoninski 5‐HT2A receptor u kompleksu sa agonistima (serotonin,lorkaserin)iantagonistima(klozapin,ziprazidon).

Dobijeni statistički i validacioni parametri za modele agonista i antagonistaukazuju na pouzdanost i dobru predviđajuću moć 3D‐QSAR modela. Najznačajnijevarijableformiranihmodeladajunamuvidunajvažnijestrukturnerazlikeizmeđunjih.Rezultati MD simulacije otkrivaju najvažnije razlike u konformacionim promenamauzrokovane vezivanjem agoniste/antagoniste, kao i interakcije liganada sa ključnimaminokiselinama, odgovornim za vezivanje. Pomoću trajektorije iz MD simulacijeizvučeni su modeli, 3D strukture 5‐HT2A receptora u njegovom aktivnom (agonist‐vezujućem)iinaktivnom(antagonist‐vezujućem)stanju.

Naosnovuovihinsilicorezultatamogućejezaključitidalijejedinjenjeagonistailiantagonista.Formiranimodelićedaljebitikorišćenizaligand‐basedistructure‐basedvirtualniskriningiracionalnidizajnnovih5‐HT2Aliganada.

390

COMBINEDLIGANDANDSTRUCTURE‐BASEDAPPROACHINSEARCH

OF5‐HT2ARECEPTORAGONISTSANDANTAGONISTS

MilicaRadan,MirjanaAntonijević,TeodoraĐikić,KatarinaNikolić,DanicaAgbaba

DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐Facultyof

Pharmacy(Serbia)

Theserotonin5‐HT2Areceptorshaveshownawiderangeofclinicalimplications

since they are involved in various physiological and pathophysiological processes.Structurally diverse ligands (agonists, antagonists, and inverse agonists) can lead todifferentbiologicalresponses,byprovokingdifferentconformationalchangesofthesereceptors.

To behave like an agonist/antagonist the molecule should have a set offunctionalgroupsandspecificinteractionswithcertainaminoacidsinthebindingsite.Understanding and explaining dissimilarities in agonist/antagonist structure andreceptorbindingwouldbebeneficialforfuturerationaldrugdesign.

Tounderstandstructuraldifferencesinpharmacophoresaswellasthebindingkinetics of agonists and antagonists, we have combined ligand‐based and structure‐based approaches. 3D‐quantitative structure‐activity relationship (3D‐QSAR) studieswereperformedonaseriesof79agonistsand90antagonists.Simultaneously,werunfourmoleculardynamics(MD)simulations:5‐HT2Aincomplexwithagonists(serotonin,lorcaserin),andantagonists(clozapineandziprasidone).

Obtained statistical and validation parameters for agonists and antagonistsmodel indicated the reliability andgoodpredictivepotential of the3D‐QSARmodels.The most influential variables of selected models gave us the insight into majorstructural dissimilarities between them. Results of MD simulation revealed majordifferencesinconformationalchangescausedbyagonist/antagonistbinding,aswellasligandinteractionswiththekeyaminoacids,responsible forthem.Additionally, fromMDsimulationtrajectory,wehaveextracted3Dstructuremodelsof5‐HT2Ainitsactive(agonist‐bound)andinactive(antagonist‐bound)state.

Using these finding we will be able to discriminate whether a compound isagonist or antagonist, in silico. Furthermore, models that we have generatedwill befurtherusedfor ligand‐basedandstructure‐basedvirtualscreeningandrationaldrugdesignofnovel5‐HT2Aligands.

391

Arh.farm 2018;68: 391-392 FHA-P10

ISPITIVANJECITOTOKSIČNEAKTIVNOSTIAMINOKISELINSKIHESTARAVITAMINAENAĆELIJAMATUMORADOJKEIPLUĆA

ŽarkoGagić1,TatjanaSrdić‐Rajić2,KatarinaNikolić3,DanicaAgbaba3

1Katedrazafarmaceutskuhemiju,UniverzitetuBanjojLuci‐Medicinskifakultet(BosnaiHercegovina),2InstitutzaonkologijuiradiologijuSrbije‐Odsekzaeksperimentalnuonkologiju,3Katedrazafarmaceutskuhemiju,

UniverzitetuBeogradu–Farmaceutskifakultet(Srbija)

Uvelikombroju studijapokazana je antitumorskaaktivnostprirodnih izomera

vitaminaE,anaročitonjihovihpolusintetskihderivata.Ciljovestudijejebioispitivanjecitotoksične aktivnosti estara α‐tokoferola sa aminokiselinama lizinom, prolinom,glutaminom,asparaginomiestaraγ‐tokotrienolasalizinom,prolinomiglutaminomnaMCF7 i MDA‐MB 231 ćelijskim linijama tumora dojke i A549 ćelijskoj liniji tumorapluća. Sve ćelijske linije tretirane su koncentracijama ispitivanih jedinjenja u opsegu0,62‐50 µM u toku 48 sati. Preživljavanje ćelija nakon tretmana ispitivanimjedinjenjima je određenoMTT‐testom.Najveći uticaj napreživljavanjemalignih ćelijasu imali α‐tokoferil lizin, α‐tokoferil asparagin u formi nitrila i γ‐tokotrienil lizin. α‐TokoferillizinjeispoljiosnažnuantitumorskuaktivnostnaA549(IC50=10,6µM)iMCF7(IC50=8,6µM)ćelijama,dokjeγ‐tokotrienillizinjejediniodispitivanihjedinjenjakojijeispoljioaktivnostnasvetrimalignećelijskelinije,saIC50vrednostima20,6µM(MCF7),28,6µM(MDA‐MB‐231)i19µM(A549).Asparaginskiestarα‐tokoferolauforminitrilajejedoveodosnažneinhibicijepreživljavanjaMDA‐MB‐231ćelija(IC50=9,2µM)kojeseodlikuju višestrukom rezistencijomna lekove koji se koriste u terapiji tumora dojke.Ispitivana jedinjenja nisu ispoljila toksičnost kaMRC‐5 zdravoj ćelijskoj liniji fetalnihfibroblastapluća.

Zahvaljujućipokazanojinvitrocitotoksičnojaktivnostiiselektivnostizamalignećelije,aminokiselinskiestriα‐tokoferolaiγ‐tokotrienolapredstavljajudobrekandidatezabudućainvivoispitivanja.

392

CYTOTOXICACTIVITYOFAMINOACIDESTERSOFVITAMINEAGAINSTBREASTANDLUNGCANCERCELLLINES

ŽarkoGagić1,TatjanaSrdić‐Rajić2,KatarinaNikolić3,DanicaAgbaba3

1DepartmentofPharmaceuticalChemistry,UniversityofBanjaLuka‐FacultyofMedicine(BosniaandHerzegovina),2DepartmentofExperimentalOncology,InstituteforOncologyandRadiologyofSerbia,3DepartmentofPharmaceutical

Chemistry,UniversityofBelgrade–FacultyofPharmacy(Serbia)

In recent studies, the antitumor activity of vitamin E derivatives has been

demonstrated. The aim of this study was to investigate the cytotoxic activity of α‐tocopherol esters with amino acids lysine, proline, glutamine, asparagine and γ‐tocotrienolesterswithlysine,prolineandglutamineonMCF7andMDA‐MB231breastcancer cell lines and A549 lung cancer cell line. All cell lines were treated withconcentrations of the test compounds in the range of 0.62‐50 μM for 48 hours. CellsurvivalaftertreatmentwiththeinvestigatedcompoundswasdeterminedbyMTTtest.

Thegreatest influenceon the survivalofmalignant cellswasobservedwithα‐tocopheryl lysine, α‐tocopheryl asparagine in the form of nitrile and γ‐tocotrienyllysine.α‐TocopheryllysineexhibitedstrongcytotoxicactivityonA549(IC50=10.6μM)andMCF7 (IC50 = 8.6 μM) cells,while γ‐tocotrienyl lysine is the only compound thatexhibited activity on all three cancer cell lines, with IC50 values of 20.6 μM (MCF7),28.6μM(MDA‐MB‐231)and19μM(A549).Theα‐tocopherylasparaginenitrileledtoastrong inhibition of the survival of MDA‐MB‐231 cells (IC50 =9.2 μM) that arecharacterizedbymultipleresistance todrugsused for treatmentofbreastcancer.Allinvestigatedcompoundsdidnotexhibit toxicitytonormalMRC‐5cell lineof the fetalfibroblastsofthelungs.

Basedontheshown invitrocytotoxicactivityandselectivityfortumorcells,α‐tocopherol and γ‐tocotrienol amino acid esters represent promising candidates forfutureinvivostudies.

393

Arh.farm 2018;68: 393-394 FHA-P11

OPTIMIZACIJAHPLCMETODEZAODREĐIVANJEMETILPARABENAIPROPILPARABENAIZHIDROGELA

MiljanJeremić,SlavicaSunarić

Katedrazafarmaciju,UniverzitetuNišu‐Medicinskifakultet(Srbija)

Parabeni su supstance koje se u farmaciji koriste kao konzervansi. Kako je

dokazanonjihovoestrogenskodelovanje,kontrolanjihoveupotrebedobijanaznačaju.Metilparabenipropilparabensusupstancesličnestrukture ipolarnosti,pa jenjihovoodređivanje HPLCmetodom zahtevno. Prisustvo ekscipijenasa i aktivnih supstanci uformulacijama dodatno otežava određivanje parabena. Cilj ovog rada je bilaoptimizacijaHPLCmetodezaodređivanjemetilparabenaipropilparabenaizhidrogela.

RastvoristandardnihsupstanciipripremljeniuzorcisupodvrgnutiHPLCanalizinaZorbaxEclipsePlusC8(3,0x150mm,3,5mm)iZorbaxEclipsePlusC18(3,0x150mm,3,5mm)kolonama.Kaomobilna faza. ispitivana je smešametanola i fosfatnogpufera(pH=2,5) i smeša metanola i vode u zapreminskim odnosima 50:50, 60:40, 70:30,80:20,90:10kao i100%metanola. Ispitivan igradijentnirežimrada.Temperatura jeodržavananakonstantnih35 ,adetekcija jevršenauUVoblastina254nm.Brzinaprotokamobilne faze je ispitivana u rasponu od 0,3 do 0,8 ml/min. Upoređivani susimetrija i širinapikova, rezolucija, retenciona vremena, selektivnost i broj teorijskihplatoazaodabranehromatografskeuslove.

Najboljiizgledpikova(simetrijaiširina),kaoirezolucijaibrojteorijskihplatoa,dobijeni su korišćenjemkolone ZorbaxEclipse PlusC18,mobilne faze procentualnogsastavametanola i fosfatnog pufera (pH=2,5) 70 prema 30 i brzine protokamobilnefaze0,5ml/min.Gradijentnirežimradasepokazaodobrimzaanalizusmešestandardametilparabena i propilparabena, međutim kod realnih uzoraka nisu dobijeni boljirezultati u odnosu na izokratni režim. Prisustvo fosfatnog pufera u mobilnoj faziznačajnopovećavakvalitethromatograma.

S obzirom na malu razliku u hemijskoj strukturi i polarnosti, HPLC analizaparabena iz farmaceutskih uzoraka predstavlja izazov. U radu smo uspešnooptimizovaliHPLCmetoduzasimultanoodređivanjemetilparabenaipropilparabenaizuzorakahidrogela.

394

OPTIMIZATIONOFTHEHPLCMETHODFORTHEDETERMINATIONOFMETHYLPARABENANDPROPYLPARABENINHYDROGELS

MiljanJeremić,SlavicaSunarić

DepartmentofPharmacy,UniversityofNiš‐FacultyofMedicine(Serbia)

Parabens are used as preservatives. Since their estrogenic activity has been

demonstrated, control over their use is gaining in importance. Methylparaben andpropylparaben are of similar structure and polarity, so their HPLC determination isdifficult.Thepresenceofinterferingsubstancesinformulationsfurthercomplicatesthedetermination.TheaimofthispaperwasoptimizationoftheHPLCconditionsforthedeterminationofmethylparabenandpropylparabenfromhydrogels.

StandardsubstancesandpreparedsamplesweresubjectedtoHPLCanalysisonZorbaxEclipsePlusC8(3.0x150mm,3.5mm)andZorbaxEclipsePlusC18(3.0x150mm,3.5mm) columns. As a mobile phase mixtures of methanol and phosphate buffer(pH=2.5)andmixturesofmethanolandwater(50:50,60:40,70:30,80:20,90:10,100:0,respectively)wereinvestigated.Gradientelutionwasalsoexamined.Thetemperaturewaskeptat35°Candthedetectionwascarriedoutat254nm.Theflowratewastestedin the range of 0.3 to 0.8ml/min. The symmetry andwidth of the peaks, resolution,retentiontime,selectivityandthenumberoftheoreticalplateswerecompared.

Thebestpeakparameters,resolutionandthenumberoftheoreticalplateswereobtainedusingZorbaxEclipsePlusC18column,amixtureofmethanolandphosphatebuffer (pH=2.5) in 70:30 volume ratio and the flow rate of 0.5ml/min. The gradientelutionwasefficientfortheanalysisofstandards,however,itwasnotadequatefortheanalysisofthesamples.Thephosphatebuffersignificantlyincreasedthequalityofthechromatograms.

Considering the small difference in chemical structure and polarity, HPLCanalysis of the parabens from pharmaceutical samples is a challenge. In this paper,HPLCconditionsforsimultaneousdeterminationofmethylparabenandpropylparabenfromhydrogelsweresuccessfullyoptimized.

395

Arh.farm 2018;68: 395-396 FHA-P12

RAZVOJHPLCMETODEZAODREĐIVANJESADRŽAJANEORGANSKIHNITRATAUIZOSORBID‐MONONITRATTABLETAMAPRIMENOM

AQBDPRISTUPA

NatašaMilović1,AnaKalinić1,JasminkaOgnjanović1,MilenaRmandić2,AnđelijaMalenović2

1Slaviamedd.o.o.,Sektorobezbeđenjaikontrolekvaliteta,2Katedrazaanalitiku

lekova,UniverzitetuBeogradu‐Farmaceutskifakultet(Srbija)

Nakvalitetfarmaceutskihsupstanciizuzetnoznačajanuticajmožeimatikoličina

prisutnihneorganskihnečistoćanaročito štonekeodnjih (npr.nitrati i nitriti)mogubiti veoma reaktivne. Cilj ovog rada bio je razvoj pouzdane, robusne, selektivne iosetljive HPLC metode za određivanje sadržaja neorganskih nitrata kao nečistoća utabletamaizosorbid‐mononitrataprimenomAQbDpristupa.

Uticaj CMPs (sadržaj metanola u mobilnoj fazi, koncentracija CH3COONH4 uvodenoj fazi, temperaturakolone)na odabraneCMAs (retencioni faktorneorganskognitrata,visinapikaibrojteoretskihplatoa)ispitanjeprimenomBoks‐Benkendizajna.Za definisanje Design Space‐a (DS), unutar koga je verovatnoća dostizanja željenihperformansi metode π≥95%, primenjene su Monte Karlo simulacije. Zbog prisustvaneorganskihnitratauplacebu,štojepotvrđenolimittestomzaidentifikacijunitratauvodi (EP 9) i HPLC analizom sa CAD detekcijom, definisana je radna koncentracijarastvoratakodanivonitratauplacebubudeispodlimitadetekcije. Ispitivanirastvorisu filtrirani kroz hidrofilne PTFE špric filtre, a eksperimenti su izvedeni na koloniNucleodur®PolarTec150mm×4,6mm,5µm,uzUVdetekciju(λ=220nm).

Veza između ispitivanih CMPs i CMAs opisana je kvadratnim matematičkimmodelom, čija je validnost potvrđena statističkim testovima. PrimenomMonte Karlosimulacija izvršeno je propagiranje greške koja potiče od izračunatih koeficijenatamodelanaodabraneodgovore,dobijena jenjihovaprediktivnadistribucija idefinisanDS. Radna tačka je odabrana iz centralnog dela DS: mobilna faza metanol‐11 mMCH3COONH4 (32:68, V/V), pH vodene faze 4,9; T=38C. Robusnost kvantitativnihperformansimetodeprocenjenajePlaket‐Burmanovimdizajnom,apogodnostmetodezadefinisanunamenupotvrđenajesprovođenjemvalidacije.

Kakosusvidobijenirezultatiuskladusadefinisanimzahtevima,potvrđenojedaje razvijenametoda pouzdana i pogodna za rutinsku kontrolu neorganskih nitrata uizosorbid‐mononitrattabletama.

396

DEVELOPMENTOFHPLCMETHODFORTHEDETERMINATIONOFINORGANICNITRATEIMPURITYINISOSORBIDE‐MONONITRATE

TABLETSBYAQBDAPPROACH

NatašaMilović1,AnaKalinić1,JasminkaOgnjanović1,MilenaRmandić2,AnđelijaMalenović2

1Slaviamedd.o.o.,QualityAssuranceandQualityControlDepartment,

2DepartmentofDrugAnalysis,UniversityofBelgrade‐FacultyofPharmacy(Serbia)

Thequalityofpharmaceuticalsubstancescansignificantlybeaffectedbypresent

inorganic impurities, especially if thesearehighly reactive (e.g.nitratesandnitrites).Theaimofpresentstudywasdevelopmentof reliable, robust, selectiveandsensitiveanalytical method for inorganic nitrate impurity assay in isosorbide‐mononitratetabletswiththeaidofAQbDapproach.

The influence of CMPs (methanol content in mobile phase, concentration ofCH3COONH4 in water phase, column temperature) on the set of selected CMAs(retentionfactorofinorganicnitrateimpurity,peakheight,numberoftheoreticalplate)was studied by Box‐Behnken design. Design space (DS)was defined byMonte CarlosimulationsasazonewhereCMAsfulfillpredefinedacceptancelimitswithahighlevelofprobabilityπ≥95%.Duetotheinorganicnitratespresenceinplacebo,confirmedbythe nitrate limit test (EP 9) and HPLC analysis with CAD detection, the workingsolutions concentration was adjusted so that the placebo nitrate level is belowdetection limit. Test solutionswere filtered through hydrophilic PTFE syringe filters,and the experiments were performed on Nucleodur® PolarTec column 150 mm×4.6mm,5μm,withUVdetection(λ=220nm).

Relationships between CMPs and CMAs were described by quadraticmathematical models, their validity confirmed statistically. Monte Carlo simulationswereusedtopropagatetheerrororiginatingfromthecalculatedmodelcoefficientstoselected responses in order to obtain their predictive distribution and to define DS.WorkingpointwasselectedfromthecentralpartofDS:mobilephasemethanol–11mMCH3COONH4 (32:68,V/V), aqueousphasepH=4.9;T=38°C.RobustnessofquantitativemethodperformancewasassessedbyPlackett‐Burmandesign,whileitssuitabilityforintendedpurposewasconfirmedbyvalidation.

Since the obtained results comply with defined requirements, reliability andsuitability of developed method for routine inorganic nitrate control in isosorbide‐mononitratetabletshasbeenconfirmed.

397

Arh.farm 2018;68: 397-398 FHA-P13 MICELIZACIJABINARNIHSMEŠAŽUČNIHSOLINATRIJUM‐

DEOKSIHOLATAINATRIJUM‐HIODEOKSIHOLATA

NemanjaTodorović1,IvanaStojanović2,VesnaTepavčević1,MihaljPoša1

1Katedrazafarmaciju,UniverzitetuNovomSadu‐Medicinskifakultet,2PharmaSwissd.o.o.(Srbija)

Žučne soli su prirodni surfaktanti, koji imaju fiziološku ulogu u emulzifikaciji

mastiudigestivnomlumenu. Ispoljavajupromotornuaktivnostutransporturazličitihlekova kroz biološke membrane, čime povećavaju njihovu bioraspoloživost. U ovomradu je ispitana micelizacija binarnih smeša žučnih soli natrijum‐deoksiholata inatrijum‐hiodeoksiholata, sa aspekta termodinamičke stabilnosti njihovih mešovitihmicela.

Napravljenisurastvorismešanatrijum‐deoksiholatainatrijum‐hiodeoksiholataurazličitimmolskimodnosima(9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9),pričemujezaizradu svih rastvora korišćena dejonizovana voda zasićena pirenom, a jonska jačinarastvorapodešena jena300mMnatrijum‐hloridom.Kritičnemicelarnekoncentracijerastvora surfaktanata određivane su spektrofluorimetrijski, praćenjem zavisnostiintenziteta prve i treće emisione trake pirena, u odnosu na ukupnu koncentracijusurfaktanata,natemperaturamaod10°C,25°C,35°Ci50°C.

Eksperimentalnoodređenevrednostikritičnihmicelarnihkoncentracijabinarnihmešovitih micela natrijum‐deoksiholata i natrijum‐hiodeoksiholata imaju niževrednosti od idealnih kritičnih micelarnih koncentracija, izračunatih po Clint‐u, štoukazuje na postojanje interakcija između različitih komponenti mešovitih micela.Koeficijent interakcije i dodatna Gibbs‐ova energija, određeni na osnovu teorijeregularnih smeša, imajunegativne vrednosti na svim ispitivanim temperaturama, štoukazujenasinergističkeinterakcijeizmeđurazličitihkomponentiumicelama,odnosnorealnemešovitemicelesutermodinamičkistabilnijeodidealnihmešovitihmicela.

Ispitivane žučne soli u binarnoj smeši stupaju u međusobne inerakcije kojefavorizujunastanakmicelauvodenomrastvoru,štozaposledicuimasniženjevrednostikritičnihmicelarnih koncentracija i termodinamičku stabilizacijumešovitihmicela. Spovećanjem temperature vrednosti kritičnih micelarnih koncentracija rastu, što seobjašnjava povećanom pokretljivošću gradivnih jedinica micela na višimtemperaturama.

398

MICELLIZATIONOFTHEBINARYMIXTURESOFBILIARYSALTSSODIUM‐DEOXYCHOLATEANDSODIUM‐HYODEOXYCHOLATE

NemanjaTodorović1,IvanaStojanović2,VesnaTepavčević1,MihaljPoša1

1DepartmentofPharmacy,UniversityofNoviSad‐FacultyofMedicine,

2PharmaSwissd.o.o.(Serbia)

Bilesaltsarenaturalsurfactantsandhaveaphysiologicalroleinemulsification

offatsindigestivelumen.Theypromotetransportofvariousdrugsthroughbiologicalmembranes, which results in the enhanced drug bioavailability. In this work, themicellization of the binary mixture of sodium‐deoxycholate and sodium‐hyodeoxycholate is examined, from the aspect of thermodynamic stability of theirmixedmicelles.

The solutions of the binary mixtures of sodium‐deoxycholate and sodium‐hyodeoxycholateindifferentmolarrations(9:1,8:2,7:3,6:4,5:5,4:6,3:7,2:8,1:9)arepreparedwith deionizedwater, saturatedwith pyrene, and the ionic strength of thesolutionsisadjustedwith300mMsodium‐chloride.Criticalmicellarconcentrationsofthesemixturesaredeterminedspectrofluorometrically,bymonitoringtheintensitiesofthe first and the third emission bands in relation to the total concentration ofsurfactants,atdifferenttemperatures:10°C,25°C,35°Cand50°C.

Experimentallydeterminedcriticalmicellarconcentrationsof thebinarymixedmicellesofsodium‐deoxycholateandsodium‐hyodeoxycholatearelowerthantheidealcritical micellar concentrations, calculated according to Clint, which indicates theexistenceofinteractionsbetweenthedifferentcomponentsofthemixedmicelles.Thecoefficient of interaction and the excess Gibbs energy, determined using the regularsolution theory, have negative values at all temperatures, indicating synergisticinteractions between the different components in micelles, that is, the real mixedmicellesarethermodynamicallymorestablethantheidealmixedmicelles.

Theexaminedbilesaltsinaqueoussolutionareinvolvedintheinteractionsthatfavourtheformationofmicelles,whichresultsinthedecreaseofthevaluesofcriticalmicellarconcentrationsandthethermodynamicstabilizationofthemixedmicelles.Byincreasing the temperature, the values of critical micellar concentrations increase,which is explained by greater mobility of the micelle's building units at highertemperatures.

399

Arh.farm 2018;68: 399-400 FHA-P14

ISPITIVANJEUSLOVAZAENANTIOSEPARACIJUMOKSIFLOKSACINAIPOTENCIJALNEHIRALNENEČISTOĆE(R,R)‐IZOMERA

MarijaMitrović,BiljanaOtašević,AnaProtić,MiraZečević

Katedrazaanalitikulekova,UniverzitetuBeogradu‐Farmaceutskifakultet

(Srbija)

Moksifloksacin‐hidrohloridustrukturiposedujedvahiralnacentra,iodmoguća

četiri izomerna oblika farmakološku aktivnost poseduje jedino (S,S)‐izomer, dok seostali izomeri smatraju potencijalnim nečistoćama. Cilj ovog rada bio je ispitivanjeuslova za razdvajanje enantiomernogpara,moksifloksacina i njegovog (R,R)‐izomera,naciklodekstrinskomtipuhiralnestacionarnefaze.

Hromatografska analiza je izvedena na Dionex UltiMate 3000 sistemu,upotrebom Chiral CD‐Ph kolone (4,6x250mm, 5µm). Mobilna faza je pripremanamešanjem acetonitrila i vodenog rastvora pufera trietilamonijum‐acetata. Protokmobilnefazebioje1mLmin‐1,talasnadužinadetekcije293nm,volumeninjektovanja10 µL. Skrining uslova za razdvajanje enantiomera moksifloksacina i (R,R)‐izomerasproveden je variranjem sadržaja acetonitrila u mobilnoj fazi (32,5‐47,5% V/V),sadržaja trietilamina (0,5‐1,5% V/V), pH vrednosti vodene faze (3,5‐4,5 podešenaglacijalnom sirćetnom kiselinom) i temperature kolone (20‐30°C) premaeksperimentalnom planu formiranom upotrebom 24 Punog faktorskog dizajna. Kaoodgovori sistemapraćeni su faktor rezolucije, faktor enantioselektivnosti i asimetrijapikova. U programu Design‐Expert® 7.0.0 izvršena je grafička obrada dobijenihrezultatakorišćenjemhalf‐normalprobabilitygrafikonaiParetodijagrama.

Kolona sa hiralnim selektorom fenilkarbamat β‐ciklodekstrinom, na kojoj jesprovedeno ispitivanje, pokazala je stereoselektivno molekulsko prepoznavanjeenantiomernogpara.Tokomeksperimentalnogradazapaženojedapovećanjesadržajatrietilaminautičenaparametrerazdvajanja,dovodećidosmanjenjafaktorarezolucijeienantioselektivnosti. Primećeno je da promena sadržaja acetonitrila dovodi dosmanjenja faktora rezolucije, dok istovremeno dolazi do poboljšanja asimetrije pika(R,R)‐izomera i povećanja faktora enantioselektivnosti. Grafičkom obradom rezultatapotvrđen je značajanuticaj svih ispitivanih faktoranaposmatraneodgovore sistema.DaljimispitivanjemdodatnojeuočenodatrietilaminipHvrednostvodenefazeimajurazličit uticaj na asimetriju pikova. Pored toga, na asimetriju (R,R)‐izomera i faktorrezolucijeznačajanuticajimaiinterakcijaovadvafaktora.

Tokom skrininga zapaženi značajni uticaji ispitivanih faktora i njihovihinterakcija na odabrane odgovore sistema, ukazuju na potrebu detaljnijih ispitivanjaposmatranihfaktoraufazioptimizacije.

400

INVESTIGATIONOFCONDITIONSFORENANTIOSEPARATIONOFMOXIFLOXACINANDITSPOTENTIALCHIRALIMPURITY(R,R)‐

ISOMERMarijaMitrović,BiljanaOtašević,AnaProtić,MiraZečević

DepartmentofDrugAnalysis,UniversityofBelgrade‐FacultyofPharmacy

(Serbia) Moxifloxacinhydrochloridecontainstwochiralcentreswhichpharmacologically

activeformis(S,S)‐isomer,whileotherisomersareconsideredaspotentialimpurities.Theaimof thisstudywasto investigateseparationconditionsofmoxifloxacinanditsenantiomer(R,R)‐isomer,usingcyclodextrinCSP.

Chromatographic analysis were performed on Dionex UltiMate 3000 systemusing Chiral CD‐Ph column (4.6x250mm, 5µm). The mobile phase was prepared bymixing acetonitrile and triethylammonium acetate buffer solution. Themobile phaseflow rate was 1 mL min‐1, detection wavelength 293 nm, injection volume 10 µL.Screening approach for separation of moxifloxacin and its (R,R)‐isomer, weremonitored by variations of acetonitrile content in mobile phase (32.5‐47.5% v/v),contentoftriethylamine(0.5‐1.5%v/v),waterphasepHvalue(3.5‐4.5setwithglacialacetic acid) and column temperature (20‐30°C) following the experimental planaccording to 24 FFD. Monitored system responses were: resolution factor,enantioselectivityfactorandassimetryofpeaks.Graphicalinterpretation(half‐normalprobability plots, Pareto charts) of results was done using Design‐Expert® 7.0.0software.

Used columnwithphenylcarbamatedβ‐cyclodextrin as chiral selector, showedenantiorecognitionofenantiomers.Duringexperimentalworkitwasobservedthattheincrease of triethylamine content has effect on separation parameters, leading todecrease of resolution and enantioselectivity factor. It was noticed that change ofacetonitrilecontentleadstodecreaseofresolutionfactor,whilesimultaneouslythereisan improvement in asymmetry of (R,R)‐isomer and increase in enantioselectivity.Graphical interpretation of results, confirmed significant effect of all investigatedfactors on system responses. Further investigation showed that triethylamine andwater phase pH value have different influence on peaks assimetry. Besides that, theinteractionofthistwofactorshavesignificantimpactonassimetryof(R,R)‐isomerandresolution.

During the screening, significant effects of the investigated factors and theirinteractions on monitored system responses, indicates the need for detailedexaminationofthisfactorsthroughoptimizationphase.

401

Arh.farm 2018;68: 401-402 FHA-P15

ISPITIVANJERETENCIONIHMEHANIZAMAAMLODIPINBESILATA,BISOPROLOLFUMARATAINJIHOVIHNEČISTOĆAUPOTREBOMTRI

RAZLIČITEHILICKOLONE

IrenaKasagić‐Vujanović1,BiljanaJančić‐Stojanović2,DarijaKnežević1,DarkoIvanović2

1Katedrazaanalitikulijekova,UniverzitetuBanjojLuci‐Medicinskifakultet(BosnaiHercegovina),2Katedrazaanalitikulekova,UniverzitetuBeogradu‐

Farmaceutskifakultet(Srbija)

Mehanizmi razdvajanja u HILIC sistemu baziraju se na specifičnim i

nespecifičnim mehanizmima, te ne postoji kvantitativan model koji može sigurnopredvidjeti ponašanje nekog jedinjenja u HILIC metodi. Vremenom je zaključeno dabrojneinterakcijemoguimativažnuuloguprilikomrazdvajanja ispitivanihjedinjenja:raspodjela/particija,adsorpcijaiprocesjonskeizmjene.HILICsistemjevrlospecifičan,razdvajanje analita uvijek se vrši sa više različitih mehanizama koji su istovremenoprisutni.Ciljovogradabiojedaseodredemehanizmirazdvajanjaispitivanihjedinjenjakorišćenjemparticionihiadsorpcionihteorijskihmodela.

Kao hromatografski sistem korišćen je Agilent 1200 sa DAD detektorom.RazdvajanjejeizvršenonatrirazličiteHILICkolone:silika,aminoidiolna,smobilnomfazom sastava ACN–50 mmol L‐1 vodeni rastvor pufera (pH 4,0 podešena sakoncentrovanom sirćetnom kiselinom), u odnosu koji je varirao od 84:16 do 96:4(V/V).UVdetekcijavršenajena230nm,zapreminainjekotovanjabilaje20μL,brzinaprotokamobilnefaze1mLmin−1itemperaturakolone30◦C.

Izračunati su regresioni koeficijenti particionih i adsorpcionih modela iodgovarajući koeficijenti determinacije (R2). Veće R2 vrijednosti imali su particionimodeli za sva ispitivana jedinjenja na amino koloni – bilo je neophodno obezbjeditiveću koncentraciju soli pufera (CH3COO‐), te na taj način suzbiti elektrostatskoodbijanjeispitivanjihjedinjenja.VećeR2vrijednostiimalisuadsorpcionimodelizasvaispitivana jedinjenja na silika i diolnoj koloni. Na sve tri kolone zaključeno je da sapovećavanjem sadržaja acetonitrila umobilnoj fazi produžava se retenciono vrijemesvih ispitivanih jedinjenja,usljedsmanjenjanjeneeluacionemoći.Zaključeno jedasenaaminokoloniretencionimehanizmisvih ispitivanih jedinjenjaprovodepostupkomrazdvajanja/particije,doknadiolnojisilikakolonimehanizmomadsorpcije.

402

INVESTIGATIONOFTHERETENTIONMECHANISMSOFAMLODIPINEBESYLATE,BISOPROLOLFUMARATEANDTHEIRIMPURITIESON

THREEDIFFERENTHILICCOLUMNS

IrenaKasagić‐Vujanović1,BiljanaJančić‐Stojanović2,DarijaKnežević1,DarkoIvanović2

1DepartmentofDrugAnalysis,UniversityofBanjaLuka‐FacultyofMedicine(BosniaandHercegovina),2DepartmentofDrugAnalysis,Universityor

Belgrade‐FacultyofPharmacy(Serbia)

Mechanisms of separation in HILIC are based on specific and non‐specific

mechanisms.There isnoquantitativemodel that cansafelypredict thebehaviorof acompound inHILICmethod. Itwasconcludedthatmany interactionscanhaveaveryimportant role during separation: partition, adsorption and process of ion exchange.HILIC system is specific, separation is simultaneously done by multiplemechanisms.Theaimof thisarticalwas todetermineretentionmechanismsof testedcompoundsusingpartitioningandadsorptionretentiontheoreticalmodels.

It'susedchromatographicsystemAgilent1200–DADdetector.SeparationswereperformedunderHILICmodeonthreedifferentcolumns:silica,aminoanddiol.MobilePhase:ACN–50mmol L‐1 aqueousbuffer solutionmixture (pH4.0was adjustedwithglacial aceticacid) in ratio from84:16 to96:4 (V/V).UVdetectionwasperformedat230nm,injectionvolume20μL,flowrate1mLmin−1,columntemperature30C.

Calculated the regression coefficients of partitioning and adsorption retentionmodels and corresponding coefficient of determination (R2). Higher values of R2 hadpartitioningmodelsforallcompoundsataminocolumn–itwasnecessarytoprovideahigher concentration of buffer salt (CH3COO‐) and to prevent the process ofelectrostatic repulsion with tested compounds. The higher value R2 had adsorptionmodelsforallcompoundsatsilicaanddiolcolumn.Onallthreecolumns,itwasshownthatwith increasingacetonitrilecontent inmobilephase, theretentiontimeof testedcompoundsextends,duetodeclineofitselutingpower.Itwasconcludedthatonaminocolumn, retention mechanisms of all tested compounds were carried out bypartitioningprocess,whileondiolandsilicacolumn,retentionmechanismsaretakingplacebyadsorptionprocess,respectively.

403

Arh.farm 2018;68: 403-404 FHA-P16

QbDPRISTUPURAZVOJUIVALIDACIJIHILICMETODEZAANALIZU

AMITRIPTILINHIDROHLORIDAINJEGOVIHNEČISTOĆA

IrenaKasagić‐Vujanović1,DarijaKnežević1,GuroForsdahl2,3,BiljanaJančić‐Stojanović3,4

1Katedrazaanalitikulijekova,UniverzitetuBanjojLuci‐Medicinskifakultet(BosnaiHercegovina),2UniverzitetuTromsø,Odsjekzafarmaciju–ArcticUniverzitetuNorveškoj(Norveška),3Laboratorijazadopingkontrolu,

SeibersdorfLaborGmbH(Austrija),4Katedrazaanalitikulekova,UniverzitetuBeogradu‐Farmaceutskifakultet(Srbija)

Quality by Design (QbD) koncept je naučno zasnovan pristup koji omogućava

definisanje prostora dizajna (DS) kao robusnog regiona u toku razvoja analitičkemetode.Ciljovogradabio jedaseprimjenomQbDkonceptarazvijeHILICmetodazakontrolukvalitetatabletasaamitriptilinom.

ProgramMODDE®Pro12.0.V.(UmetricsSartorius‐Stedim,Švedska)korišćen jezakreiranjeplanaeksperimenta,statističkuobradurezultata, tezakreiranjeprostoraznanja i prostora dizajna. Kao tečni hromatogram korišćen je Agilent 1200 sa DADdetektorom, kolona ZORBAX‐NH2 (250 mm x 4,6 mm, 5 μm veličina čestica),mobilnafaza: acetonitril–60 mM rastvor amonijum acetata (pH 4,5 podešenakoncentrovanom sirćetnomkiselinom) u odnosu 92,5:7,5 (V/V). Temperatura kolone30°C,protokmobilnefaze1mLmin‐1,talasnadužinaodređivanja254nm.

Kroznekolikodobrodefinisanihkoraka,QbDkonceptkorišćenjezarazvojprveHILICmetodezaanalizuamitriptilinainjegovihnečistoća.Sciljemdasepostignedobrorazdvajanje spitivanih jedinjenja sa što kraćim vremenom trajanja analize, za procesoptimizacije korišćen je Box‐Behnken dizajn, a kao kritični parametri korišćeni su:sadržaj acetonitrila u mobilnoj fazi, pH vrijednost vodenog rastvora pufera ikoncentracija amonijum‐acetata. Procjena robusnosti metode izvršena je upotrebomfrakcionogfaktorskogdizajna(FFD24‐1),azatimsuispitaniiostaliparametrivalidacijemetode:selektivnost,linearnost,tačnost,preciznostiodređenisulimitdetekcije(LOD)ilimitkvantifikacije(LOQ).

Upotrebom QbD koncepta razvijena je i validirana metoda za određivanjeamitriptilinhidrohloridainjegovihnečistoćaA,B,CiFutabletama.Ovakavpristupurazvojumetodeomogućiojeugrađivanjekvalitetautokuprocesarazvojametode,tejerazvijenarobusnametoda.Metodajevalidiranaiprimjenjenazaanalizuamitriptilinainjegovihnečistoćautabletama.

404

QbDORIENTEDDEVELOPMENTANDVALIDATIONOFTHEHILIC

METHODFORTHEANALYSISOFAMITRIPTYLINEHYDROCHLORIDEANDITSIMPURITIES

IrenaKasagić‐Vujanović1,DarijaKnežević1,GuroForsdahl2,3,

BiljanaJančić‐Stojanović3,4

1DepartmentofDrugAnalysis,UniversityofBanjaLuka‐FacultyofMedicine(BosniaandHerzegovina),2DepartmentofPharmacy,UniversityofTromsø‐TheArcticUniversityofNorway(Norway),3DopingControlLaboratory,

SeibersdorfLaborGmbH(Austria),4DepartmentofDrugAnalysis,UniversityorBelgrade‐FacultyofPharmacy(Serbia)

AQualitybyDesign(QbD)approach isascientificbasedconceptwhichcanbe

usedtodefineDesignSpace(DS)foranalyticalmethods.TheaimofthispaperwastodeveloptheHILICmethod for thequalitycontrolofamitriptyline tabletsusingaQbDconcept.

MODDE®Pro12.0.V.(UmetricsSartorius‐Stedim,Sweden)wasusedtocreateanexperimentplan,statisticalanalysisofresultsandconstructingaknowledgespaceanddesignspace.LiquidchromatographAgilent1200serieswithDADdetector,ZORBAX‐NH2column(250mmx4.6mm,5μmparticlesize),mobilephase:acetonitrile‐60mMaqueous ammonium acetate solution, pH 4.5 adjusted with concentrated acetic acid(92.5:7.5V/V).Columntemperature:30°C,mobilephaseflow1mLmin‐1,wavelengthofdetection254nm.

Through several well defined steps, a QbD concept was implemented fordevelopmentof the firstHILICmethod foraminotriptilineand its impuritiesanalysis.Toreachthedesiredchromatographicresolutionwithalimitednumberofexperimentsin a minimum amount of time, Box‐Behnken design was used to simultaneouslyoptimize for important chromatographic parameters: the acetonitrile content in themobilephase,pHoftheaqueousphaseandtheconcentrationofammoniumacetateinaqueous phase. Using the fractional factorialdesign (FFD 24‐1), the robustness of themethodwasestimated,andthenotherparametersofthevalidationofthemethodweredetermined:selectivity,linearity,accuracy,precision,LODandLOQ.

UsingtheQbDconcept,aHILICmethodfortheanalysisofamitriptylineanditsimpuritiesA,B,CandF in tabletswasdevelopedandvalidated.Thisapproach in thedevelopment of the method has enabled the embedding of quality during thedevelopmentofthemethodandarobustmethodhasbeendeveloped.Themethodwasvalidatedandappliedtotheanalysisofamitriptylinehydrochlorideanditsimpuritiesintablets.

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Arh.farm 2018;68: 405-406 FHA-P17 ODREĐIVANJETELMISARTANAINJEGOVIHNEČISTOĆAU

TABLETAMARP‐HPLCMETODOMSGRADIJENTNIMELUIRANJEM

DraganaVukadinović1,VladimirDobričić2,AleksandraAranđelović1,DanijelaRadojičić1,JelenaMaksić1,

BiljanaJančić‐Stojanović3,OliveraČudina2

1Odeljenjezaispitivanjeikontrolulekova,Službazafarmaceutskudelatnost,Vojnomedicinskaakademija,2Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet,3Katedrazaanalitikulekova,Univerzitetu

Beogradu‐Farmaceutskifakultet(Srbija)

Telmisartan jenepeptidniantagonistaangiotenzin II receptorakoji sekoristiuterapiji hipertenzije. Cilj ovog rada je razvoj i validacija nove RP‐HPLC metode zakvalitativnuikvantitativnuanalizutelmisartanainjegovihnečistoćaAiCutabletama.

OptimalnorazdvajanjeizmeđuispitivanihsupstancisasličnimpKavrednostimapostignuto je pomoću Chromolith C18 kolone (100 mm x 4,6 mm, 5 μm veličinačestica),natemperaturiod25°Ciuzgradijentnoeluiranje.KaoeluentAupotrebljenje0,1%vodenirastvortrifluorosirćetnekiseline,dokjekaoeluentBkorišćenacetonitril.Volumen injektovanja bio je 20 μL, a UV detekcija vršena je na 230 nm. Metoda jevalidiranaiispitanisusledećiparametrivalidacije:selektivnost,linearnost,preciznost,tačnost,limitdetekcije(LOD)ilimitkvantifikacije(LOQ).PredloženaRP‐HPLCmetodajeprimenjenazakvalitativnuikvantitativnuanalizutelmisartanainečistoćaAiCufilmtabletama.

Na retencionim vremenima koja odgovaraju telmisartanu i njegovimnečistoćama nisu uočene komponente koje interferiraju. Lineranost metode jeodređenauopsegukoncentracija1‐15μgmL‐1 za telmisartan (r=0,9999),0,12‐1,50μgmL‐1 za nečistoću A (r = 0,9998), odnosno 0,11‐1,50 μg mL‐1za nečistoću C (r =0,9997). Preciznost metode dokazana je na osnovu izračunatih RSD vrednosti zatelmisartan (0,99%), nečistoću A (1,22%) i nečistoću C (1,37%). Tačnost metodepotvrđena je Recovery vrednostima za telmisartan (99,8%), nečistoću A (99,3%) inečistoćuC(99,4%).OsetljivostmetodepokazanajedefinisanjemLODiLOQvrednostizanečistoće.Rezultati ispitivanjaufilmtabletamaodgovarajuzahtevimaspecifikacije,sadržajtelmisartanaje99,35%,sadržajnečistoćeAjemanjiodLOQ,anečistoćeCmanjiodLODvrednosti.

PredloženaRP‐HPLCmetodapoddatimeksperimentalnimuslovimapredstavljabrz, precizan, tačan i selektivanpostupak za istovremenukvalitativnu i kvantitativnuanalizutelmisartanainečistoćautabletama.

406

DETERMINATIONOFTELMISARTANANDITSIMPURITESINTABLETSBYRP‐HPLCMETHODWITHGRADIENTELUTION

DraganaVukadinović1,VladimirDobričić2,

AleksandraAranđelović1,DanijelaRadojičić1,JelenaMaksić1,BiljanaJančić‐Stojanović3,OliveraČudina2

1DepartmentofDrugControlandExamination,ServiceforPharmaceutical

Activity,MilitaryMedicalAcademy,2DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐FacultyofPharmacy,3DepartmentofDrugAnalysis,

UniversityofBelgrade‐FacultyofPharmacy(Serbia)

Telmisartan is a non‐peptide angiotensin II receptor antagonist withantihypertensiveproperty.TheaimofthisworkwasdevelopmentandvalidationofanewRP‐HPLCmethod for qualitative and quantitative analysis of telmisartan and itsimpuritiesAandCintablets.

Theoptimalseparationamong investigatedsubstanceswithsimilarpKavalueswasachievedusingChromolithC18column(100mmx4.6mm,5μmparticlesize),attemperature of 25°C and gradient elution. As the eluentA, 0.1%aqueous solution oftrifluoroacetic acid was used, whereas acetonitrile was used as the eluent B. Theinjectionvolumewas20μlandUVdetectionwasperformedat230nm.Themethodwas validated in terms of selectivity, linearity, precision, accuracy, limit of detectionand limitofquantification.TheproposedRP‐HPLCmethodwasapplied inqualitativeandquantitativeanalysisoftelmisartanandimpuritiesinfilmcoatingtablets.

At retention times corresponding to telmisartan and its impurities nointerferenceswereobserved.Linearitywasprovedinconcentrationranges1‐15μgmL‐1fortelmisartan(r=0.9999),0.12‐1.50μgmL‐1forimpurityA(r=9998)and0.11‐1.50μgmL‐1forimpurityC(r=0.9997).TheprecisionofthemethodwasprovedaccordingtocalculatedRSDvalues for telmisartan(0.99%), impurityA(1.22%)and impurityC(1.37%).Theaccuracyof themethodwasconfirmedbyRecoveryvaluesobtained fortelmisartan(99.8%),impurityA(99.3%)andimpurityC(99.4%).Thesensitivityofthemethod was shown by defining LOD and LOQ values of impurities. The results ofanalysisoffilmcoatingtabletsmeettherequirementsofthespecification.Thecontentoftelmisartanwas99.35%,imputityAwasbelowLOQ,whilethecontentofimpurityCwasbelowLODvalue.

TheestablishedRP‐HPLCmethodwas found tobe rapid,precise, accurateandselective for qualitative and quantitative analysis of telmisartan and its impurities infilmcoatingtablets.

407

Arh.farm 2018;68: 407-408 FHA-P18

KARAKTERIZACIJABIOMOLEKULASAANTIBIOTIČKIMDEJSTVOMIZENDOFITAPHOMOPSISSPECIES

JankoIgnjatović1,NevenaMaljurić1,JelenaGolubović2,MilošPetković3,

MatjažRavnikar4,BorutŠtrukelj4,BiljanaOtašević1

1Katedrazaanalitikulekova,UniverzitetuBeogradu‐Farmaceutskifakultet(Srbija),2InstitutJožefŠtefan(Slovenia),3Katedrazaorganskuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet(Srbija),4Katedrazafarmaceutskubiologiju,UniverzitetuLjubljani‐Farmaceutskifakultet

(Slovenija)

Evolucionarno, biljke su razvile određene odbrambene sposobnosti kao što je

proizvodnja specifičnih sekundarnih metabolita kako bi se zaštitile od insekata i/iliživotinja koje se hrane njima. Povrh toga, tkiva biljaka sadrže mnoge vrstemikroorganizamakojesenazivajuendofiti.Endofitisuspeci icnizasvakogdomacinaikarakteristicni su za klimatske uslove i geografsku oblast u kojoj se biljka domacinrazvija. Postoje brojni izveštaji o različitim biološkim aktivnostima endofitnih gljiva.OdreditiantibiotičkipotencijalendofitaPhomopsisspecieskojarastenačetinarimauSloveniji.

Kulturaendofitajezasejananaagarpločamaiostavljenanasobnojtemperaturiza proces rasta, nakon čega je metanolni ekstrakt endofita (50%, v/v) podvrgnutmikrodilucijskom testu. Zapažena je značajna inhibicija rasta Gram (‐) bakterijeEscherichia Coli. Ekstrakt je dodatno analiziran pomocu semipreparativne HPLCmetode s ciljem otkrivanja, razdvajanja i sakupljanja frakcija vrhova pripisanihspecifičnimbiomolekulama.Dodatnojeodređenanjihovahemijskastrukturauzpomocmasne spektroskopije visoke definicije (LC‐QTOF analiza) i spektroskopije nuklearnemagnetnerezonance.

Dvaglavnavrhakojaseeluirajunaretencionomvremenuod3,34i3,89minuta,pokazale su molekularne jone u režimu pozitivne elektrospejne jonizacije sa m/zvrednostimaod318i335,respektivno.Njihovidentitetjekonačnoopisankao(Z)‐(Z)‐2‐acetoksiprop‐1‐en‐1‐il‐3‐(3‐((E)‐3,4‐dihidroksipent‐1‐en‐1‐il)‐oksiran‐2‐il)‐akrilat i(Z)‐(Z)‐2‐acetoksiprop‐1‐en‐1‐il‐3‐(3‐((E)‐4‐hidroksi‐3‐oksipent‐1‐en‐l‐il)oksiran‐2‐il)‐akrilat).Predlozena jedinjenjakaopotencijalniterapeutskiagensisemogukoristitikao vodeci molekuli za otkrivanje novih antibiotika. Takođe, kombinovane analiticketehnikekorisceneuovomistrazivanjumoglebisekoristitikaoanalitičkaplatformazakarakterizacijuiodređivanjenovihmolekulskihentitetauekstraktimaendofita.

408

CHARACTERIZATIONOFBIOMOLECULESWITHANTIBIOTIC

ACTIVITYFROMENDOPHYTEPHOMOPSISSPECIES

JankoIgnjatović1,NevenaMaljurić1,JelenaGolubović2,MilošPetković3,MatjažRavnikar4,BorutŠtrukelj4,BiljanaOtašević1

1DepartmentofDrugAnalysis,UniversityofBelgrade‐FacultyofPharmacy(Serbia),2JožefStefanInstitute(Slovenia),3DepartmentofOrganicChemistry,

UniversityofBelgrade‐FacultyofPharmacy(Serbia),4ChairforPharmaceuticalBiology,UniversityofLjubljana‐FacultyofPharmacy

(Slovenia)

Evolutionally, plants have developed certain defending capabilities such as

producingofspecific secondarymetabolites inorder torepell insectsand/oranimalsthat are feeding on them. Moreover, tissues of plants contain many types ofmicroorganismswhicharerefferedtoasendophytes.Endophytesarespecificforeachhostandarecharacteristicfortheconditionsandgeographicalareainwhichthehostdevelops.Atthesametime,therearenumerousreportsondifferentbiologicalactivitiesofendophyticfunghi.AntibioticpotentialofendophytePhomopsisspeciesgrowingonconifersinSloveniahasbeentested.

Crude matherial was sawed on agar plates and left on room temperature forgrowing process upon which its methanol extract (50%, v/v) was subjected tomicrodilution assay. Considerable inhibition of the growth of Gram(‐) bacteriaEscherichia Coli was noticed. Prepared sample was further investigated usingsemipreparative HPLC in order to detect, separate and collect fractions of peaksattributedtospecificbiomoleculesandconsequentlyelucidatetheirchemicalstructurewith the assistance of high definition mass spectroscopy (LC‐QTOF analysis) andnuclearmagneticresonancespectroscopy.

Two major peaks which eluted at retention times at 3.34 min and 3.89 minshowedmolecular ions in positive electrospay ionisationmodewithm/z values 318and335,respectively.Theiridentitywasfinalycharacterizedas(Z)‐(Z)‐2‐acetoxyprop‐1‐en‐1‐yl‐3‐(3‐((E)‐3,4‐dihydroxypent‐1‐en‐1‐yl)oxiran‐2‐yl)‐acrilate and (Z)‐(Z)‐2‐acetoxyprop‐1‐en‐1‐yl‐3‐(3‐((E)‐4‐hydroxy‐3‐oxopent‐1‐en‐1‐yl)oxiran‐2‐yl)‐acrylate).

Beside proposition of presented compounds as potential therapeutic agents,these structures could even be used as leading molecules for novel antibioticsdiscovery.Moreover, combined analytical techninquesused in this research couldbeemployedasanalyticalplathformforcharacterizationanddeterminationofmolecularentitiesinendophyteextracts.

409

Arh.farm 2018;68: 409 FHA-P19 ADESIRABILITYBASEDMULTIOBJECTIVEAPPROACHFOR

MODELING,PREDICTINGANDOPTIMIZATIONOFEXPERIMENTALCONDITIONFORFORCEDDEGRADATIONOFROSUVASTATIN

MajaHadzievaGigovska1,AnaPetkovska1,JelenaAcevska2,NatalijaNakov2,BlagicaManchevska1,PackaAntovska1,

SonjaUgarković1,AnetaDimitrovska2

1Research&Development,ALKALOIDAD,2University„SsCyrilandMethodius”‐FacultyofPharmacy,Skopje(Macedonia)

Forced degradation studies provide data to support identification of possible

degradants, degradation path ways, intrinsic stability and validation of stabilityindicating methods. Generally, a trial and error approach (adopted to select thestrength, temperatureand timeof exposure toachievedesireddegradation) involvesconsiderable cost, time consumption and scientific expertise with high incidence ofrandomresults.Theaimofthisresearchwastousedesirabilityfunctionasasystematicapproachformultiobjectiveoptimizationoftheexperimentalconditionforconductingforceddegradationonrosuvastatinasmodeldrug.

The degradation behavior of rosuvastatin was estimated based on 23 fullfactorial designs, where strength of HCL/NaOH/H2O2, temperature and time ofexposure were considered as independent and % total impurities (determined byvalidated RP‐HPLC method) as dependent variable. The experimental results wereevaluatedusingtheproposedpredictivemodelwhichincludeddesirabilityfunctiontofindsetofcoordinateswithdesirabilityvaluecloseto1.

Statistical evaluation, using ANOVA, showed that proposed model fit the datawell and have high predictive power for new observations. Selections of optimalconditionswereperformedusingdesirabilityfunction,wheretargeteddegradationwasset in the range 5‐20% and% ofmajor unknown impurity to 0.1%. The predictedconditionsbythemodelwithhighdesirabilityvaluewere0,1MHCl/25oC/45minutesand0.1MNaOH/25oC/45minutes,respectably.Theseconditionswereselectedintheverificationstudy,andtheobtainedresultsdemonstratedgoodagreementbetweentheexperimentaldataandpredictedvalues.

The proposedmodel presents an efficient and easily accomplishable approachfor searching optimum degradation conditionswhich can replace the trial and errorapproach,providinginformationrichpresentationofresultswithlesslaboratorywork.

410

Arh.farm 2018;68: 410 FHA-P20 DOEAPPROACHFOROPTIMIZATIONOFAGENERICHIGH‐PERFORMANCELIQUIDCHROMATOGRAPHYMETHODFOR

DETERMINATIONOFUNDECLAREDCOMMONCOUGHANDCOLDINGREDIENTSINNATURALPRODUCTS

MarijaZafirova,JelenaAcevska,LiljanaUgrinova,

GabrielaPetrovska‐Dimitrievska,VasilKarchev,NatalijaNakov,KaterinaBrezovska,AnetaDimitrovska,SuzanaTrajkovic‐Jolevska

Ss.CyrilandMethodiusUniversity‐FacultyofPharmacy,Skopje(Macedonia)

Inaneedofaddressingthecurrentworryingproblemoffalsifiedmedicines,an

all‐encompassingmethodthatcanbeusedforscreeningandquantifyingcomponentsofdubious samples ofmedicines and detecting undeclared active substances in naturalproductscanbeofgreatuse.

The aim of this work was to develop a fast and simple, generic HPLC‐DADmethodforsimultaneousdeterminationofthemostfrequentlyusedactivesubstancesin cough and cold preparations,whichwould be applicable for detecting undeclaredingredientsinnaturalproductsforcoughandcoldtreatment.

The chromatographic separation was achieved on an Agilent 1260 Infinitysystem, on a Poroshell 120 EC‐C8 50 mm×4.6 mm, 2,7 μm column, using gradientelutionconsistedofsolventA(0,1%formicacidinwater)andsolventB(0,1%formicacidinacetonitrile).

Elevencommoncoughandcoldactiveingredientswithbroadrangeofpolaritieswere successfully separated on a reversed phase core‐shell HPLC column usinggradient elution with a very simple mobile phase in just 14 minutes with excellentsensitivity and good resolution. The optimisation was performed by the design ofexperimentsapproachusingtheCentralCompositeFaceCentered(CCF)modelintwosets of experiments. The summed results of both sets of experiments suggest theproposed method conditions providing satisfactory resolution between all of thecritical peak pairs. The proposed method has been validated according to ICHguidelinesandprovedtobesuitableforthesimultaneousqualitativeandquantitativedeterminationoftheselectedcompounds.

TheoptimizedefficientandfastHPLCmethodforsimultaneousdeterminationofthemostfrequentlyusedcoughandcoldactivesubstancesisgenerallyapplicableforanumberofpossible formulation compositionsof coughand coldmedicines, includingpreparationswheretheyshouldnotbepresent.

411

Arh.farm 2018;68: 411-412 FHA-P21 PRIMENAMETODAMULTIVARIJANTNIHANALIZAGLAVNIHKOMPONENTIIHIJERARHIJSKOGGRUPISANJAUISPITIVANJU

RAZDVAJANJAJEDINJENJAZIPRASIDONATEČNOMHROMATOGRAFIJOM

MarijaČarapić1,KatarinaNikolić2,AdamSmolinski3,DanicaAgbaba2

1AgencijazalekoveimedicinskasredstvaSrbije,2Katedrazafarmaceutsku

hemiju,UniverzitetuBeogradu‐Farmaceutskifakultet(Srbija),3CentralMiningInstitute,DepartmentofEnergySavingandAirProtection,Katowice(Poland)

Ziprasidon je novi atipični antipsihotik druge generacije, koji deluje kaoantagonista na serotoninskim i dopaminskim receptorima, i inhibira preuzimanjenorepinefrina.Glavniciljhemometrijskestudijejeispitivanjeselektivnosti20reverzno‐faznih (RP) stacionarnih faza u odnosu na ziprazidon i šest nečistoća ((I‐V) inepoznata).VelikastrukturnasličnostziprasidonainečistoćeIIbilajeključniproblemirazdvajanjekritičnogparajebiloodlučujućezaodabirRPstacionarnefaze.

Za analizu glavnih komponenti (PCA) korišćen je matematički program SoftIndependent Modeling of Class Analogy SIMCA P+ 12.0, a za analizu hijerarhijskoggrupisanja(HCA)korišćenjeprogramMATLABver.6.5.

Ispitivanje selektivnosti 20 RP stacionarnih faza je vršeno pri dobijenimoptimalnimeksperimentalnimuslovima(25ºC;pH:2,5;cTEA:1%icKH2PO4:50mM)uodnosu na ispitivana jedinjenja. Eksperimentalno dobijeni hromatografski parametri(brojteoretskihplatoa‐N,faktorsimetrijepika‐SF,rezolucija‐Rsifaktorselektivnosti‐α između jedinjenja koja se blizu eluiraju) na 20 RP stacionarnih faza analizirani suprimenomPCAiHCAanaliza.

Grafikoni rezultata iPCAkoeficijenatavariablizaPC1(osnovnakomponeta1) iPC2(osnovnakomponeta2)pokazujuglavnerazlikeizmeđusvih20RP‐kolonaiglavnesličnosti između hromatografskih parametara. Rs i α su odabrani kao najznačajnijiparametrizaizborstacionarnefaze.Dobijenidendogramipokazalisutriglavnegrupezastacionarnefazeičetirigrupezahromatografskeparametre.DendogramiRPkolonai hromatografskih parametara su prikazani i obojenom mapom što je jednostavnijametodavizuelizacije.

Određene su RP stacionarne faze posebno selektivne za efikasno razdvajanjeziprasidonaistrukturnosličnihjedinjenjaprimenomPCAiHCA(grupa1PCAigrupaCAHC):Waters Spherisorb® ODS1, Waters Spherisorb® ODS2 i Nucleosil® 100‐5 C18.Dobijeni podaci su u skladu sa eksperimentalnim rezultatima. Odabrana je WatersSpherisorb®ODS1kolonazaHPLCmetoduuodnosunanajboljuSFi faktorretencije(k’).

412

MODELINGOFLIQUIDCHROMATOGRAPHYSEPARATIONOF

ZIPRASIDONECOMPOUNDSUSINGMULTIVARIATEMETHODSOFPRINCIPALCOMPONENTANDHIERARCHICALCLUSTERING

ANALYSIS

MarijaČarapić1,KatarinaNikolić2,AdamSmolinski3,DanicaAgbaba2

1MedicinesandMedicalDevicesAgencyofSerbia,2DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐FacultyofPharmacy(Serbia),3CentralMiningInstitute,DepartmentofEnergySavingandAir

Protection,Katowice(Poland)

Ziprasidone is a novel „atypical” or „second generation” antipsychotic drug

whichactsprimarilythroughserotonergicanddopaminergicreceptorantagonism,andas an inhibitor of the norepinephrine reuptake. The main aims of the presentedchemometric study was to test selectivity of the set of 20 Reversed‐phase (RP) ‐columns towards ziprasidone and its six impurities ((I‐V) and one unknown).Separation of structurally similar pair of ziprasidone/impurity II caused analyticalproblemsandwasdecisivefortheselectionofthesuitableRP‐column.

ThePrincipalComponentAnalysis(PCA)forcolumnclassificationwasdonewithuseofSIMCAP+12.0programandHierarchicalClusteringAnalysis(HCA)withuseofMATLABver.6.5.withadditionalalgorithms.

The obtained optimal chromatographic conditions (25C, pH 2,5, cTEA 1% andcKH2PO4 50 mM) were used to test a set of 20 RP‐columns. Plate numbers (N),symmetry factors (SF), resolution (Rs) and selectivity (α) of investigated compoundswere subjected toPCAandHCAanalysis. Scoreplot and loadingplotsPC1 (principalcomponent1)vsPC2(principalcomponent2)visualizethemaindifferencesbetweenall 20 RP‐columns and main similarities between hromatographic parameters,respectively. The Rs and α were selected as the most significant for the columnselection.TheobtaineddendrogramsrevealthreedistinctclustersofRP‐columnsandfour clusters of chromatographic parameters. The color map of data was used as asimpler presentation of the dendrograms of RP‐columns and chromatographicparameters.

The RP‐columns selective for the efficient separation of ziprasidone and itsstructurallyrelatedcompoundsweredefinedbyPCAandHCA(group1 inPCAstudyand same cluster C in HCA study) : Waters Spherisorb® ODS1, Waters Spherisorb®ODS2andNucleosil®100‐5C18.The resultswere inaccordancewithexperimentallyobtained results. Finally, the columnWaters Spherisorb®ODS1was selected for theHPLCmethodduetobestSFandretentionfactor(k’).

413

Arh.farm 2018;68: 413-414 FHA-P22 ELEKTROHEMIJSKAKARAKTERIZACIJAREDOKSPROCESA

BRIMONIDINAIVARENIKLINA

ValentinaRadulović1,MaraAleksić2,VeraKapetanović1,DanicaAgbaba3

1Katedrazaanalitičkuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet,2Katedrazafizičkuhemijuiinstrumentalnemetode,UniverzitetuBeogradu‐

Farmaceutskifakultet,3Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet(Srbija)

Brimonidin(BRIM),agonistα2‐adrenergičkogreceptora,primenjujeselokalnou

terapiji glaukoma oka, a vareniklin (VAR), parcijalni agonist nikotinskih receptora, uterapiji odvikavanja od pušenja. Oba leka su derivati hinoksalina, ali različitogfarmakoterapijskog dejstva. Cilj je bio proučiti parametre redoks procesa iokarakterisatielektrohemijskoponašanjeBRIMiVAR.

VAR je proučavan cikličnom voltametrijom (CV) uz elektrodu od staklastogugljenika (GCE). Za elektrohemijsko proučavanje BRIM primenjena je polarografijajednosmerne struje (DCP) i kapljuća živina elektroda (DME). Korišćen jepotenciostat/galvanostatμAUTOLAB(Ecochemie,Holandija),Metrohm663VAStandiinterfejsIME663(Metrohm,Švajcarska)podržaniGPES4.9programom,itroelektrodnaćelija.

Redukcija BRIM proučavana je:primenomDCP/DME, pripH=2,0‐12,0 (Briton‐Robinsonov pufer); a predstavljena je polarografskim talasom (Ic) sa pomerajempolutalasnihpotencijalakanegativnijimvrednostima(E1/2=‐0,060pH‐0,167;R=0,9993).Pri pH≤3,0 uočen je slabo izražen drugi redukcioni talas (IIc). Logaritamska analizaBRIMtalasa(Ic)ukiselojsredinidalajevrednostim(brojaprotona)1,76‐1,84.

PrimenomCV/GCE,urazličitimpuferimapH2,15‐9,20ipripotencijalu‐1,5Vdo+1,5V,zabeleženisuVARpikovi:katodni(Ic)udirektnomidvaanodnapika(Ia,IIa)upovratnom smeru. Zabeleženo je pomeranje potencijala Ic i Ia pika ka negativnijimvrednostima sa porastom pH (Ep,Ic=‐0,052pH‐0,340; R=0,9976 i Ep,Ia=‐0,053pH‐0,274;R=0,9972). Kod drugog anodnog pika IIa prisutno je pomeranje potencijala pika kamanje pozitivnim vrednostima (Ep,IIa= ‐0,061pH+0,533;R=0,9750), što odražavaolakšanu oksidaciju. Na osnovu dobijenih rezultata│Ep,Ia–Ep1/2,Ia│odnosno│Ep,IIa–Ep1/2,IIa│;zatimIp,Ic/Ip,Ia~1iΔEp~60mV;utvrđenjebrojrazmenjenihe‐.

Redukcija BRIModvija se na C=N vezi hinoksalina do1,4‐dihidro‐BRIMputemdvoelektronskog procesa i učešće 2H+. U jako kiseloj sredini, uz utrošak još dvaelektrona,nagrađeni1,4‐dihidro‐BRIMsedaljeredukujedo1,2,3,4‐tetrahidro‐BRIM.Uzučešće2e‐/2H+,odvijaseredukcijaVARnaC=Nvezihinoksalinado1,4‐dihidro‐VAR,astepen reverzibilnosti raste sa porastom pH. Drugi anodni pik (IIa) VAR odgovaraireverzibilnoj oksidaciji produkta redukcije (1,4‐dihidro‐VAR) uz učešće 1e‐/1H+,donastajanja2‐hidroksi‐1,4‐dihidro‐VAR.

414

ELECTROCHEMICALCHARACTERISATIONOFBRIMONIDINEANDVARENICLINEREDOXPROCESSES

ValentinaRadulović1,MaraAleksić2,VeraKapetanović1,DanicaAgbaba3

1DepartmentofAnalyticalChemistry,UniversityofBelgrade‐FacultyofPharmacy,2DepartmentofPhysicalChemistryandInstrumentalMethods,

UniversityofBelgrade‐FacultyofPharmacy,3DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐FacultyofPharmacy(Serbia)

Brimonidine(BRIM)isanα2‐adrenergicreceptoragonistusedinlocaltherapyof

glaucoma. Varenicline (VAR), a partial agonist of nicotinic receptors, is applied forsmoking cessation. Both drugs are quinoxaline derivatives with differentpharmacotherapeutic effects. The aimwas to point out parameters of redox processanddesribeelectrochemicalbehaviourofBRIMandVAR.

VARwasexaminedbyCyclicVoltammetry(CV)/GlassyCarbonElectrode(GCE),while Direct Current Polarography (DCP)/Dropping Mercury Electrode (DME) wereusedforBRIMelectrochemicalstudy.Potentiostat/galvanostatμAUTOLAB(Ecochemie,Netherlands),Metrohm663VAStandand IME663(Metrohm,Switzerland)supportedbyGPES4.9,andsystemofthreeelectrodeswereemployed.

Reduction of BRIM was investigated by DCP/DME in Britton‐Robinson bufferpH=2.0‐12.0andpolarographicwave (Ic)withchangesofhalfwavepotential towardsmorenegativevalues(E1/2=‐0.060pH‐0.167;R=0.9993)wasobtained.AtpH≤3.0,poorlydifferentiated the second reductionwave (IIc)wasnoticed. Logarithmic analysis of Icwaveinacidicmediaresultedinexperimentalvaluesofm(H+‐number)=1.76‐1.84.

ApplicationofCV/GCEandpotential range ‐1.5V to+1.5V (indifferentbufferspH=2.15‐9.20) for VAR study gave: cathodic peak (Ic)(direct scan) and two anodicpeaks (Ia, IIa) (reverse scan).Movement of Ic and Ia peak potential with pH increasetowardsmorenegativevalueswasrecorded(Ep,Ic=‐0,052pH‐0,340;R=0,9976andEp,Ia=‐0,053pH‐0,274;R=0,9972).Moving of potential anodic IIa peak towards less positivevalues (Ep,IIa= ‐0,061pH+0,533;R=0,9750) reflects facilitated oxidation. Based onobtainedresultsfor│Ep,Ia–Ep1/2,Ia│and│Ep,IIa–Ep1/2,IIa│;alsoIp,Ic/Ip,Ia~1andΔEp~60mV;numberoftransferrede‐wasestablished.

BRIM reduction appears at C=N bond of quinoxaline by participation of 2e‐/2H+,forming1,4‐dihydro‐BRIM.Instrongacidicmedia,additional2e‐reductionstepofformed1,4‐dihydro‐BRIM is registered giving 1,2,3,4‐tetrahydro‐BRIM.VAR reductionhappens with 2e‐/2H+ participation, at C=N bond of quinoxaline, generating 1,4‐dihydro‐VAR.Reversibility increaseswith increasingpH.Thesecondanodic (IIa)VARpeakcorresponds to irreversible1e‐/1H+oxidationof reductionproduct (1,4‐dihydro‐VAR)forming2‐hydroxy‐1,4‐dihydro‐VAR.

415

Arh.farm 2018;68: 415-416 FHA-P23

ODREĐIVANJESADRŽAJAEFEDRIN‐HIDROHLORIDAU

FARMACEUTSKIMPREPARATIMAPRIMENOMRPHPLCMETODE

BrankaIvković1,MilkicaCrevarSakač1,JelenaPurić2,MilicaTasić1

1Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet,2Zdravstvenaustanova‐apoteka„ZeroPharm“,ogranak„Primavera“,

Beograd(Srbija)

Efedrin spada u grupu „mešovitih” simpatomimetika koji svoj efekat ostvarujudirektno preko adrenergičkih receptora i indirektno preko transportera za ponovnopreuzimanjekateholamina.Uterapijisekoristikaovazokonstriktorukapimazanos.Uovom radu razvijena je i validiranaRP‐HPLCmetodaza određivanje sadržaja efedrin‐hidrohlorida u farmaceutskim doziranim oblicima. Hromatografsko razdvajanje jepostignutonaZORBAXExtendC18,250x4,6mm,5μmkolonisamobilnomfazomkojučini smeša 0,1% H3PO4 i acetonitrila (90:10, v/v), pri brzini protoka od 1 ml/minidetekciji na 210 nm. Validacija je izvedena u skladu sa ICH smernicama. Metoda jelinearnauopsegukoncentracijaod20do80μg/mL(r=0,9995).Preciznostmetode jeispitananadvanivoa:ponovljivostmetode i srednjapreciznost.Naosnovudobijenihvrednosti relativne standardne devijacije za ponovljivost (1,22 % < 2%) i srednjupreciznost(1,09%<3%)možesezaključitida jemetodaprecizna.Tačnostmetodejeispitana za tri koncentracije (80, 100 i 120%u odnosu na radnu koncentraciju).Dobijene Recovery vrednosti za sve tri ispitivane koncentracije su u intervalu od98,17% do 101,38% što zadovoljava zahteve za tačnost metode (98‐102%). Malepromene hromatografskih parametara (radne temperature kolone i sastava mobilnefaze)nisuznačajnouticalenaispitivanehromatografskeparametrečimejepotvrđenarobusnostmetode.ValidiranaRP‐HPLCmetodajeprimenjenazaodređivanjesadržajaefedrin‐hidrohlorida u kapima za nazalnu primenu Dobijeni rezultati za sadržajefedrina(99,02do99,36%)zadovoljavajuzahteveod90%do110%.Validiranametodajeekonomična,efikasna,tačnaipreciznapasemožeprimenjivatiurutinskojkontrolipreparatakojisadržeefedrin.

416

DEVELOPMENTANDVALIDATIONOFRPHPLCMETHODFORDETERMINATIONOFEPHEDRINEHYDROCHLORIDEIN

PHARMACEUTICALDOSAGEFORM BrankaIvković1,MilkicaCrevarSakač1,JelenaPurić2,MilicaTasić1

1DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐Facultyof

Pharmacy,2HealthInstitution‐Pharmacy„ZeroPharm“,Department„Primavera“,Belgrade(Serbia)

In this paper development of new RP HPLC method for determination ofephedrine hydrochloride in pharmaceutical dosage forms was described. HPLCseparation was performed on ZORBAX Extend C18, 250 x 4,6 mm, 5μmchromatographic column. Column temperature was set on 25 °C. Mobile phaseconsistedof0.1%H3PO4andacetonitril(90:10,v/v).Flowrateofmobilephasewas1.0mL/minanddetectionwavelength210nm.Inequilibratedchromatographicsystem5μLodpreparedsolutionswasinjected.Thedevelopedmethodwasvalidatedaccordingto ICH guidelines. Method is linear over the concentrations range from 19,7 to 78.9μg/mL (r=0.9995). Precision was tested at two levels: intra‐assay precision andintermediateprecision.Calculatedrelativestandarddeviations(1.22%<2%forintra‐assay precision and 1.09% < 3% for intermediate precision) confirmed thatmethodwas precise. Accuracywas tested at three concentrations levels (80, 100 and 120%)and confirmed by calculated recovery values (98.17% to 101.38% which is inaccordance with the requirements 98% to 102%). Small variations of mobile phasecompositionandcolumntemperaturedidnotaffectqualitativeandquantitativesystemresponsessignificantly,whichprovedmethod'srobustness.ValidatedRP‐HPLCmethodwasappliedforassaydeterminationofephedrinehydrochloridein0.5%and1.0%nosedrops. Assay was calculated by calibration curve method and working standardmethod.Resultsobtained(99.36%in0.5%nosedropsand99.02%in1.0%nosedropsbycalibrationcurvemethodand99.40%in0,5%nosedropsand99.00%in1.0%nosedrops by working standard method) were in accordance with requirements ofmanufacturer.

417

Arh.farm 2018;68: 417-418 FHA-P24

ODREĐIVANJESADRŽAJAPARTENOLIDAUFARMACEUTSKIM

PREPARATIMAPRIMENOMRPHPLCMETODE

BrankaIvković1,MilkicaCrevarSakač1,BiljanaFidanovski2,DraganaStanković1

1Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu‐Farmaceutski

fakultet,2Zdravstvenaustanova‐apoteka„ZeroPharm”,ogranak„Primavera“,Beograd(Srbija)

Partenolid je seskviterpenski lakton germakranolidnog tipa. Predstavlja

modifikaciju germakranolida, jedinjenja koje nastaje iz ciklodekadienskog katjona,germakradiena. Izolovan je iz biljkeTanacetumparthenium (povratić). Elektrofilni α‐metilen‐γ‐laktonski prsten lako interaguje sa nukleofilnim (tiolne i amino grupe)delovima bioloških molekula, a prisustvo epoksidne grupe u molekulu intenziviraaktivnost laktona. Najnovija istraživanja pokazuju da partenolid pored antimikrobne,antiinflamatorneiantiproliferativnepokazujeiantimigrenoznuaktivnost.Pokazanojedafitopreparatipovratićasmanjuju:frekvencumigrenoznihbolova,jačinumigrenoznihbolova, stepenmučnine i povraćanja. Imajući u vidu navedene podatke razvijena je ivalidirana RP‐HPLC metodaza određivanje sadržaja partenolida u farmaceutskimdoziranim oblicima koji sadrže ekstrakt povratića. Kao stacionarna faza korišćena jeZORBAXExtendC18,250x4,6mm,5μmkolona,amobilnufazučinilajesmešavodeiacetonitrilačijiseodnosmenjatokomvremenaprikonstantnojbrziniprotokamobilnefaze od 1 ml/min. Detekcija je vršena na 210 nm. Metoda je linearna u opsegukoncentracijaod0,030do1,20mg/mL(r=1,0000).Preciznostmetodejeispitananadvanivoa:ponovljivostmetodeisrednjapreciznost.NaosnovudobijenihvrednostiRSDzaponovljivost(0,9997%<2%)isrednjupreciznost(1,0358%<3%)možesezaključitida metoda ispunjava zahteve za preciznost. Tačnost je ispitana za tri koncentracije(80%,100%i120%).DobijeneRecoveryvrednostizasvetriispitivanekoncentracijesuu intervaluod99,84%do100,47%što ispunjavazahteve tačnostimetode(98‐102%).Validirana RP‐HPLC metoda je primenjena za određivanje sadržaja partenolida ukapsulama koje sadrže ekstrakt povratića. Dobijeni rezultati za sadržaj partenolida(96%)ispunilisuzahteveod90do110%.

418

DEVELOPMENTANDVALIDATIONOFRPHPLCMETHODFORDETERMINATIONOFPARTENOLIDEINPHARMACEUTICALDOSAGE

FORM BrankaIvković1,MilkicaCrevarSakač1,BiljanaFidanovski2,

DraganaStanković1

1DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐FacultyofPharmacy,2HealthInstitution‐Pharmacy„ZeroPharm“,Department

„Primavera“,Belgrade(Serbia)

Partenolid is a sesquiterpenic lactone of the germacranolid type. It represents

the modification of germacranolide, a compound formed from cyclodecadiengermacradiene which is isolated from the plant Tanacetum parthenium. Theelectrophilic α‐methylene‐γ‐lactone ring easily interacts with nucleophilic (thiol andaminogroups)partsofbiologicalmolecules,andthepresenceoftheepoxygroupinthemolecule intensifies the activity of the lactone. Recent studies have shown thatpartenolide,inadditiontoantimicrobial,antiinflammatoryandantiproliferativeagents,showsantimigrenousactivity.Ithasbeenshownthatpreparationsofthisplantreduce:the frequencyofmigrainepain, theseverityofmigrainepain, thedegreeofvomittingandsickness.DevelopmentandvalidationofnewRPHPLCmethodfordeterminationofpartenolide in pharmaceutical dosage forms has been described. ChromatographiccolumnZORBAXExtendC18,250x4.6mm,5μmwasusedforanalysisofinvestigatedcompound. Analysis was performed at room temperature whichmeans that columntemperature was not controled. Gradient elution was used. Ratio of water andacetonitrile, which were components of mobile phase, changed over time (0 to 5minutes–10%ofacetonitrile,5do20minutes‐shareofacetonitrilehasgrownto35%,20 do 30 min – isocratic elution with 35% of acetonitrile, 30 to 40 min ‐ share ofacetonitrile has grown to 50%). Flow rate of mobile phase was 1.0 mL/min anddetectionwasperformedat210nm.Injectedvolumeofpreparedsolutionwas5μL.ThedevelopedmethodwasvalidatedaccordingtoICHguidelines.Methodislinearovertheconcentrations range from0.030 to 1.20mg/mL (r=1,0000). Precisionwas tested attwo levels: intra‐assay precision and intermediate precision. Calculated relativestandarddeviations(0.9997%<2%forintra‐assayprecisionand1.0358%<3%forintermediateprecision) confirmedprecisionofmethod.Accuracywas tested at threeconcentrationslevels(80,100and120%)andconfirmedbycalculatedrecoveryvalues(99.84% to 100.47%) which corresponds with the requirements (98% to 102%).ValidatedRP‐HPLCmethodwasapplied fordeterminationofpartenolide in capsules.Assay was calculated by calibration curve method and working standard method.Results obtained (96.26% by calibration curve method and 96.02% by workingstandardmethod)wereinaccordancewithrequirementsofmanufacturer.

419

Arh.farm 2018;68: 419 FHA-P25

EQUIVALENCETESTSASANEWSTATISTICALAPPROACHIN

ANALYTICALMETHODTRANSFERS

AleksandraPetrovska,MarijaVelickovska,NatasaAnevskaStojanovska,SonjaUgarkovic

InstituteforResearchandDevelopment,AlkaloidAD‐Skopje(Macedonia)

Thetransferofananalyticalmethodfroma laboratory,where itwasoriginally

developed and validated, to another laboratory, which is close to an additionalproductionsiteaswellasthetransferforoutsourcingpurposesbecameanimportantissue during the life cycle of a product. With respect to the method performancecriteria, all guidelines agree to evaluate accuracy and precision (variability) duringanalytical method transfer. For both, statistical equivalence tests are preferred oversignificance tests such as t‐test or F‐test, recommended by ISPE concept andUSP<1010>Generalchapter.Forthepurposesofourstudy,wehave implementedthemethod described by Wätzig in order to compare the means obtained in the twolaboratoriesusinganequivalencetest.First,wehavedenotedtestedparameter(µR‐µS)by θ, and the nominal reference value by θo.We have defined symmetrical toleranceintervalaroundθo,as[θo‐ε,θo+ε].100·(1‐2ɑ)confidenceintervalδL,CLandδH,CLforµR‐µSare calculated with ɑ=0.05.Equivalence can be proved if the whole confidenceinterval is included within interval of tolerance (δL,TOL≤δL,CL)˄(δH,CL≤δH,TOL), so nullhypothesis (H0=θ≤θo‐ε)˄(θ≥θo+ε) is rejected andalternativehypothesis (H1=θO‐ε <θ<θO+ε)isaccepted.

Analyticalmethodtransfersonseveralpharmaceuticalformswereinvestigatedinthisstudy.TheobtainedresultswerecomparedbyusingF‐testandequivalencetestby Wätzig. Set up of tolerance intervals depends mostly form method parameter,specification limit,definedbiasbetween two labs, etc. From thedataobtained, ithasbeenconfirmed thatF‐test isnot suitablefor comparison,mainlywherethevalues forvariancesweresmall.Regardingtheequivalencetestapplied,itwasprovedthatthereisnodifferencebetween two laboratories.Usingequivalence tests is theapproachofchoice,becausesystematicandrandomerrorcanbefollowedbybothlaboratories.

420

Arh.farm 2018;68: 420-421 FHA-P26

ODREĐIVANJESADRŽAJAKOFEINAUKOZMETIČKIMPREPARATIMA

ZAKOSUPRIMENOMEKSTRAKCIJENAČVRSTOJFAZIIHPLCMETODE

KristinaMladenov,SlavicaSunarić

Katedrazafarmaciju,UniverzitetauNišu‐Medicinskifakultet(Srbija)

Unovijevreme,nanašemtržištususveprisutnijikozmetičkipreparatizakosu

sa kofeinom namenjeni prevenciji i tretmanu alopecije. Kofein je često aktivnakomponenta šampona, jer stimuliše rast kose i poboljšavamikrocirkulaciju kože. Oninhibira enzimsku aktivnost 5‐α‐reduktaze i fosfodiesteraze i omogućava ponovnouspostavljanjeciklusarastakose.Preporučenakoncentracijakofeinaušamponimajeuopsegu1‐3%.Kozmetičkipreparatitipašamponasuuzorcisloženoghemijskogsastavainemogusedirektnoanaliziratibezprethodnoizvršeneadekvatnepripremeuzoraka.Ciljovogradabiojeprimenametodeekstrakcijenačvrstojfazizapripremušamponaza HPLC analizu kofeina. Pored toga, proveravan je i sadržaj kofeina u kozmetičkimpreparatimazakosunanašemtržištu.

Šamponisadeklarisanimkofeinomanabavljenisuulokalnimapotekama.Tačnakoncentracijakofeinanijenavedenanadeklaraciji.Analiziranasudvapreparata:Baleašampon(Dm‐drogeriemarkt,Karlsruhe,Germany)iAlpecin®C1(DrKurtWolff,Bielefeld,Germany).Uzorci su rastvoreni u 10mLdestilovane vode i pripremljeni korišćenjemekstrakcije na čvrstoj fazi. Najbolji rezultati dobijeni su prečišćavanjem uzorka iekstrakcijomkofeinaizšamponaprimenomChromabond®HR‐X(100mg,1mLvolume,Macherey‐Nagel,Germany).Reverzno‐faznaHPLCanaliza izvršena jenakoloniRestekUltraIBDC18(3µm,150×3mm),amobilnafazasastojalaseodmetanolaivode(40:60,V/V). Sadržaj kofeina u šamponuBalea je (1,016 ± 0,08) g/100g (1,016%), dok je upreparatuAlpecin®C1bio (1,08±0,2)g/100g (1,08%), što jeu skladusaočekivanimvrednostimailiteraturnimpodacima.

Rezultati pokazuju da su analizirani kozmetički preparati dobrog kvaliteta upogledu sadržaja kozmetički aktivne supstance. Samim tim, pokazano je i da sepredloženipostupakpripremeuzorakaiusloviekstrakcijenačvrstojfazizaekstrakcijukofeina iz šampona kao i primenjeni uslovi HPLC analize, mogu koristiti u kontrolikvalitetaovihkomercijalnihpreparata.

421

ASSESSMENTOFCAFFEINECONTENTINHAIRCAREPRODUCTSBY

SOLIDPHASEEXTRACTIONANDHPLCMETHOD

KristinaMladenov,SlavicaSunarić

DepartmentofPharmacy,UniversityofNiš‐FacultyofMedicine(Serbia)

On our market, hair products containing caffeine have recently been used to

prevent and treat alopecia. Caffeine is an active ingredient in shampoos, because itstimulates hair growth and improves skinmicrocirculation. It inhibits 5‐α‐reductaseand phosphodiesterases and allows a restoration of the hair growth cycle. Therecommendedcaffeineconcentrationinshampoosisintherangefrom1‐3%.Giventhecomplexityoftheshampooasanextractionmedium,inordertofurtheranalyzeit,anappropriatesamplepreparationhastobeperformedpreviously.ThegoalofthispaperwastodevelopthesolidphaseextractionmethodforHPLCdeterminationofcaffeineinshampoos.Inaddition,thecaffeinecontentinhaircarepreparationswasalsochecked.

Caffeine‐enriched shampoos were purchased at local pharmacies. The precisecaffeinecontentwasnotdeclared.Twoproductswereanalyzed:BaleaShampoo(Dm‐drogerie markt, Karlsruhe, Germany) and Alpecin® C1 (Dr Kurt Volff, Bielefeld,Germany). Samples were dissolved in 10 mL of distilled water. Further samplepreparationwasdoneusingsolid‐phaseextractionChromabond®HR‐Xcartridges(100mg, 1 mL volume, Macherei‐Nagel, Germany) which gave the best results regardingsample clean‐up and caffeine extraction. The reverse‐phase HPLC analysis wasperformedusingRestekUltraIBDC18column(3µm,150×3mm),andmethanol‐watersolution (40:60, v/v), asamobilephase.Foundcontentof caffeine inBalea shampooand Alpecin® C1 was (1.016 ± 0.08) g/100 g (1.016%) and (1.08 ± 0.2) g/100 g(1.08%), respectively. These results agreed with the expected values and literaturedata.

Resultsofthisworkshowthatexaminedproductshavegoodqualityintermsofactive ingredient content. The proposed extraction method and applied HPLCdetermination was suitable for the analysis of caffeine in shampoos, therefore theycouldbeusedinqualitycontrolofthesecommercialpreparations.

422

Arh.farm 2018;68: 422-423 FHA-P27

VALIDACIJAHPLC‐UVMETODEZAODREĐIVANJE

MOKSIFLOKSACINAICIPROFLOKSACINAUPERITONEALNOJTEČNOSTIKODPACIJENATANAPERITONEALNOJDIJALIZI

MajaKoraćević1,2,PredragDžodić1,

RadmilaVeličkovićRadovanović1,3,TatjanaCvetković3,4

1Katedrazafarmaciju,UniverzitetuNišu‐Medicinskifakultet,2UniverzitetuNišu‐Inovacionicentar,3Klinikazanefrologiju,KliničkicentarNiš,4Institutza

biohemiju,UniverzitetuNišu‐Medicinskifakultet(Srbija)

NovaHPLC‐UVmetoda,kombinovana saprecipitacijomproteina, jenamenjena

zakvantifikacijumoksifloksacina i ciprofloksacinauperitonealnoj tečnosti pacijenatasa peritonitisom, koji su na lečenju kontinuiranom ambulatornom peritonealnomdijalizom (CAPD).Precipitacija proteina iz uzoraka peritonealne tečnosti je izvršenapomoćuledene0,1%trifluorosirćetnekiselineumetanolu(v/v).UanalizijekorišćenaAgilentZorbaxSB‐C18analitičkakolona(150mmx4,6mm;3,5µm).Optimalniuslovizahromatografskorazdvajanjesu:mobilnafazametanol–0,1%trifluorosirćetnakiselina(34:66,v/v),protokod1ml/minitemperaturakolone35°C,uzUVdetekcijuna285nm.Ukupno trajanje hromatografskog rana iznosi oko 10min. Tokom validacijemetode,linearnost je potvrđena u koncentracionom opsegu 0,2‐50 µg/ml, sa koeficijentomkorelacijevećimod0,9857zaobaanalita.Preciznostmetodeutokujednogiutokuvišedanajezadovoljavajuća,sarelativnomstandardnomdevijacijomnižomod13,92%,doktačnostmetodeobuhvatavrednostiuopsegu87,76‐111,74%,zaobaanalita.Dobijenirezultati ukazuju da je primenjenametoda jednostavna, precizna, brza i pogodna zarazdvajanje i kvantifikacijumoksifloksacina i ciprofloksacina u peritonealnoj tečnostikod pacijenata na CAPD. Stoga, metoda može da se primeni u optimizaciji dozemoksifloksacinailiciprofloksacinauterapijiperitonitisa.

423

VALIDATIONOFHPLC‐UVMETHODFORDETERMINATIONOFMOXIFLOXACINANDCIPROFLOXACININPERITONEALFLUIDOF

PATIENTSONPERITONEALDIALYSIS

MajaKoraćević1,2,PredragDžodić1,RadmilaVeličkovićRadovanović1,3,TatjanaCvetković3,4

1DepartmentofPharmacy,UniversityofNiš‐FacultyofMedicine,2UniversityofNiš‐Innovationcenter,3ClinicofNephrology,ClinicalCenterNiš,4Instituteof

Biochemistry,UniversityofNiš‐FacultyofMedicine(Serbia)

New HPLC‐UV method, combined with protein precipitation, is developed for

quantification of moxifloxacin and ciprofloxacin in peritoneal fluid of patients withperitonitis, who are on continuous ambulatory peritoneal dialysis (CAPD)treatment.Precipitationofproteinsfromperitonealfluidsampleswasperformedwithice‐cold0.1%trifluoroaceticacid inmethanol (v/v).AgilentZorbaxSB‐C18analyticalcolumn (150 mm x 4.6 mm; 3.5 µm) was used. The optimal conditions forchromatographicseparationwere:mobilephasemethanol–0.1%trifluoroaceticacid(34:66,v/v),flowrateof1ml/min,andcolumntemperatureof35°C,withUVdetectionwavelength at 285 nm. The total run time was around 10 min. During validation,linearitywas confirmed over the concentration range 0.2‐50 µg/ml,with correlationcoefficientshigher than0,9857 forbothanalytes.Theassayexhibitedadequate intra‐dayand inter‐dayprecision,withrelativestandarddeviation less than13.92%,whilethe accuracy ranged 87.76‐111.74%, for both analytes.The obtained results indicatethat the method is simple, precise, fast and suitable for the separation andquantification ofmoxifloxacin and ciprofloxacin in the peritoneal fluid of patients onCAPD. Therefore, the proposed method has a potential to be applied for theoptimisationofdosageduringperitonitistherapywithmoxifloxacinorciprofloxacin.

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Arh.farm 2018;68: 424-425 FHA-P28

UTICAJMICELARAZLIČITOGNAELEKTRISANJAKAOSIMULIRAJUĆIH

SISTEMABIOMEMBRANANAJONIZACIJURUPATADINA

MarijaPopovićNikolić1,GordanaPopović2,KristinaStojilković2,MajaDobrosavljević2,DanicaAgbaba1

1Katedrazafarmaceutskuhemiju,UniverzitetuBeogradu‐Farmaceutskifakultet,2Katedrazaopštuineorganskuhemiju,UniverzitetuBeogradu‐

Farmaceutskifakultet(Srbija)

Rupatadin je selektivni antagonist histaminskih H1 receptora druge generacijekojiseprimenjujeuterapijialergijskogrinitisa ihroničneurtikarije.Rupatadinsadržitri jonizaciona centra, dva aromatična amina i jedan cikličan alkilamin. Doziranifarmaceutskioblicizaoralnuprimenukaoaktivnusupstancusadržerupatadinfumarat.Urastvorurupatadinfumaratauspostavljasesložensistemprotolitičkihravnotežakojiuključuje tri bazna centra rupatadina i dve karboksilne grupe fumarne kiseline.PoznavanjepKavrednostilekovaneophodnojezaprocenufarmakokinetičkihosobinaibioraspoloživosti.UfiziološkimuslovimapKavrednostimogubitipromenjeneuodnosuna vodeni rastvor usled interakcija sa naelektrisanim i polarnim biomolekulima.Ispitivanje jonizacije u pojednostavljenim sistemima biomembrana, kao što sumicelarni rastvori surfaktanata, pruža bolji uvid u ponašanje lekova u fiziološkimuslovima.Ispitanjeuticajmicelarnihrastvora,anjonskognatrijum‐dodecilsulfata(SDS),katjonskog cetiltrimetilamonijum‐bromida (CTAB) i nejonskog 4‐oktilfenolpolietoksilata(TX‐100)naprotolitičkeravnotežerupatadina.

Potenciometrijski su određene pKa vrednosti bez i u prisustvu 10‐2 Msurfaktanata, na temperaturi 25oC i pri konstantnoj jonskoj sili (0,1 M NaCl).Potenciometrijski podaci analizirani su primenom programa Hyperquad. Nezavisnoodređene pKa vrednosti fumarne kiseline korišćene su kao ulazni parametri zaodređivanjepKavrednostirupatadina.

Određene su pKa vrednosti u vodenom rastvoru (pKa1=3,34; pKa2=4,72;pKa3=6,75)iuočenjeuticajsvihprimenjenihsurfaktanata,SDS(∆pKado+1,44);CTAB(∆pKa od ‐1,99 do +0,14); TX‐100 (∆pKa od ‐0,72 do +0,38), na promenu jonizacije.Jonizujućicentrirupatadinauključujuseuelektrostatičke,hidrofobne,dipolinterakcijei vodonične veze sa micelama. Dijagram raspodele ravnotežnih oblika u funkciji pHukazuje da je promena raspodele najizraženija u pH oblasti 4 – 8 koja obuhvatabiofarmaceutskiznačajnepHvrednosti.

Pomeranje protolitičkih ravnoteža rupatadina pod uticajemmicela ukazuje dabiomolekuli različite polarnosti i naelektrisanja u fiziološkim uslovimamogu izazvatipromenuraspodeleravnotežnihoblikaodkojihzaviserastvorljivostipermeabilnost.

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THEEFFECTSOFDIFFERENTLYCHARGEDMICELLESASBIOMEMBRANEMIMETICSYSTEMSONTHEIONIZATIONOF

RUPATADINE

MarijaPopovićNikolić1,GordanaPopović2,KristinaStojilković2,MajaDobrosavljević2,DanicaAgbaba1

1DepartmentofPharmaceuticalChemistry,UniversityofBelgrade‐FacultyofPharmacy,2DepartmentofGeneralandInorganicChemistry,Universityof

Belgrade‐FacultyofPharmacy(Serbia) Rupatadine belongs to selective second‐generation histamine H1 receptors

antagonists,usedinseasonalallergicrhinitisandchronicurticaria.Rupatadinecontainsthree ionizable basic centers, two aromatic and one cyclic aliphatic amine.Pharmaceutical dosage forms contain rupatadine fumarate as an active substance.Complexsystemofprotolyticequilibriaestablishesinsolutionofrupatadinefumarateincludingthreerupatadinebasiccentersandtwocarboxylicgroupsoffumaricacid.ThepKa values are necessary for estimation of pharmacokinetic properties andbioavailabilityofdrugs.Under thephysiologicalconditionsprotolyticequilibriacouldbeshiftedduetointeractionswithbiomolecules.Theinvestigationsofionizationinthepresent of simplified biomembrane systems (micellar solutions of surfactants) givebetter insight into physiological drug behavior. The effects of surfactants, sodiumdodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and 4‐octylphenolpolyethoxylates(TX‐100)onrupatadineionizationhavebeeninvestigated.

The pKa valueswere determined potentiometrically in the absence and in thepresence of 10‐2 M surfactants at 25°C and constant ionic strength (0.1 M NaCl).PotentiometricdatawereanalyzedinprogramHyperquad.IndependentlydeterminedpKa values of fumaric acid were used as input parameters for determination ofrupatadinepKavalues.

Theionizationinwaterwasdefined(pKa1=3.34;pKa2=4.72;pKa3=6.75)andshiftinprotolyticequilibriainthepresenceofmicelles,SDS(∆pKaupto+1.44);CTAB(∆pKafrom‐1.99to+0.14);TX‐100(∆pKafrom‐0.72to+0.38),wereobserved.Theionizationcenters of rupatadine are involved in electrostatic, hydrophobic, dipole interactions,and hydrogen bonds with micelles. Distribution diagram of equilibrium forms as afunctionofpHindicatesthatchangeinionizationisthemostexpressedinpHrange4–8whichincludesbiopharmaceuticallyimportantpHvalues.

The shift in rupatadine protolytic equilibria indicates that biomolecules ofdifferent charge and polarity could change distribution of equilibrium formsresponsibleforsolubilityandpermeability.