DERIVATIVE SPECTROPHOTOMETRIC METHOD FOR DEYERMINATION OF ACYCLOVIR IN POLYMERIC NANOPARTICLES

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1 Universidde de São Pulo Bibliotec Digitl d Produção Intelectul - BDPI Deprtmento de Frmáci - FCF/FBF Artigos e Mteriis de Revists Científics - FCF/FBF 2012 DERIVATIVE SPECTROPHOTOMETRIC METHOD FOR DEYERMINATION OF ACYCLOVIR IN POLYMERIC NANOPARTICLES QUIMICA NOVA, SAO PAULO, v. 35, n. 1, pp , SEP-OCT, Downloded from: Bibliotec Digitl d Produção Intelectul - BDPI, Universidde de São Pulo

2 Quim. Nov, Vol. 35, No. 1, , 2012 DERIVATIVE SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF ACYCLOVIR IN POLYMERIC NANOPARTICLES Guilherme Diniz Tvres*, Gisele Miki Ishikw, Tlit Ferreir Monteiro, Crolin Znolini, Érik Ros Mri Kedor- Hckmnn, Nádi Arci Bou-Chcr e Vldi Olg Consiglieri Fculdde de Ciêncis Frmcêutics, Universidde de São Pulo, Av. Prof. Lineu Prestes, 580, São Pulo - SP, Brsil Recebido em 28/2/11; ceito em 13/6/11; publicdo n web em 8/8/11 Not Técnic A derivtive spectrophotometric method ws vlidted for quntifiction of cyclovir in poly (n-butylcynocrylte) (PBCA) nnoprticles. Specificity, linerity, precision, ccurcy, recovery, detection (LOD) nd quntifiction (LOQ) limits were estblished for method vlidtion. First-derivtive t nm eliminted interferences from nnoprticle ingredients nd presented linerity for cyclovir concentrtions rnging from 1.25 to 40.0 µg/ml (r = ). Precision nd ccurcy dt demonstrted good reproducibility. Recovery rnged from 99.3 to LOD ws 0.08 µg/ml nd LOQ, 0.25 µg/ml. Thus, the proposed method proved to be esy, low cost, nd ccurte, nd therefore, n useful lterntive to quntify cyclovir in nnoprticles. Keywords: cyclovir; polymeric nnoprticles; derivtive spectrophotometry. INTRODUCTION In recent decdes, there hs been considerble interest in the reserch of drug delivery using prticulte systems s crriers for smll nd lrge molecules. Accordingly, nnoprticles hve been used s physicl pproch to modify nd improve the phrmcokinetic nd phrmcodynmic properties of lrge number of drugs. 1 Nnocrriers re currently being investigted for mny therpeutic pplictions to overcome typicl drug delivery chllenges, such s incresing physicochemicl stbility, enhncing cellulr uptke of poorly permeble molecules, reducing cellulr nd tissue clernce of drugs, s well s sustining drug delivery. 2,3 So, the therpeutic efficcy nd sfety of drugs cn be significntly improved by trgeted delivery using nnoprticles. 4 Nowdys, much ttention hs been pid to the poly(lkylcyno- -crylte) (PACA) nnoprticles due to their ese of preprtion, low toxicity nd biodegrdbility. Thus, these systems re lredy employed in clinicl development for cncer therpy. 5 After orl dministrtion, PACA nnoprticles cn protect the ctive molecules nd control their relese by n erosion mechnism. 6 On the other hnd, the surfce modifiction of these nnocrriers using polymers with specific properties hs been reported to be promising wy to improve the orl biovilbility of drugs. 7 Thereby, studies hve estblished tht prtilly quternized derivtive of chitosn, N,N,N- -trimethylchitosn (TMC), cn be used s n bsorption enhncer for lrge nd/or hydrophilic compounds cross intestinl epithelium TMC cts minly by opening the tight junctions between epithelil cells to llow the prcellulr trnsport of these compounds. 11 As consequence, recent nnoprticle systems exhibit new nlyticl chllenges becuse of the wide vriety of compounds in their composition nd lso, the need to detect smller mounts of drugs. A significnt number of techniques, specilly spectrophotometry nd high-performnce liquid chromtogrphy (HPLC), re described for the quntifiction of drugs in PACA nnoprticles. Conventionl spectrophotometric methods re very susceptible to the presence of interferences in complex mixtures such s nnoprticules, requiring prior tretment or extrction of the nlyte, nd not lwys *e-mil: diniztvres@usp.br providing dequte response. Despite the undoubted dvntges of using HPLC technique, this one hs some limittions such s the high cost of instrumenttion nd opertion, reltively long time for nlysis nd the need of experience in hndling the equipment nd processing smples. In ddition, the disdvntges relted to the use of solvents such s dischrge, opertor helth nd environmentl dmge encourge the development of simpler nd fster methods, with smller mounts of solvents nd without prior extrction steps. In this sense, derivtive spectrophotometric method with detection in UV is low cost nd is esily pplied to nlysis of rw mterils nd phrmceuticls, s well s in kinetic studies of drug relese, detection of nlyte in the presence of decomposition compounds, nd mny other pplictions. 18 Derivtive spectrophotometry (DS) is bsed on so clled derivtive spectr which re generted from conventionl or zero-order scn. The derivtiztion of zero-order spectrum cn led to the seprtion of overlpped signls nd elimintion of bckground cused by presence of other compounds in smple without previous seprtion or purifiction. Nowdys, this technique is considered very useful dditionl tool which helps to solve vrious nlyticl problems. Severl pplictions hve been found in mny res of nlysis, especilly in phrmceuticl Some methods employing DS were reported to the quntifiction of cyclovir (9-[2-hydroxyethoxymethyl]-9H-gunine, ACV) (Figure 1) in order to quntify this drug in phrmceuticl dosge forms, such s injections nd crems 22 or in the presence of gunine, its min impurity. 23 Nevertheless, ccording to our knowledge, there re no reports concerning the quntifiction of ACV in PACA nnoprticles by this technique. In this sense, the im of this work ws to purpose rpid nd effective first-order spectrophotometric method to quntify ACV in Figure 1. Chemicl structure of ACV

3 204 Tvres et l. Quim. Nov empiricl formultion contining poly(n-butylcynocrylte) (PBCA) nnoprticles coted or uncoted with TMC. EXPERIMENTAL Apprtus nd conditions A Shimdzu UV-1601 double-bem spectrophotometer with 1.0 cm qurtz cells nd dt processing cpcity ws used. The first-order derivtive (zero-crossing method) spectr were obtined over the nm rnge, Δλ = 2.0 nm, scling fctor = 1.0 nd mplitude = ± 0.1. The determintions were mde t nm ( 1 D ). Methnol:Acetonitrile (MeOH:ACN) (8:2 v/v) ws used s solvent mixture. Chemicls Acyclovir reference stndrd (~99% purity) ws kindly supplied by Aché Phrmceuticls Lbortories (São Pulo, SP, Brzil). Acyclovir rw mteril of phrmceuticl grde ws kindly donted by Medley Phrmceuticl Industry (Cmpins, SP, Brzil). The monomer n-butylcynocrylte ws purchsed from B. Brun Lbortories (Tuttlingen, Bden-Württemberg, Germny). Chitosn (low moleculr weight, 92% decetylted, viscosity cps) nd Dextrn were obtined from Sigm Chemicl Compny (St Louis, MO, USA). Methyl iodide ws purchsed from Merck (Drmstdt, Hessen, Germny). All other chemicls nd solvents used were of nlyticl grde. N,N,N-trimethylchitosn ws synthesized by reductive methyltion of chitosn bsed on previously described method 24 nd 1 H-nucler mgnetic resonnce spectr ws obtined (Figure 1S, supplementry mteril). Nnoprticles preprtion Synthesis of PBCA nnoprticles Poly(n-butylcynocrylte) nnoprticles (PBCA) were obtined by emulsion polymeriztion ccording to the method described by Couvreur nd Vuthier 25 with some modifictions: n-butylcynocrylte monomer (1.0%) ws dded dropwise under mgnetic stirring (800 rpm) into 10.0 ml of queous solution (ph 2.5), contining Dextrn 70 (1.0%). After 4 h, bout µl of 0.1 M NOH ws dded, until neutrliztion of colloidl suspension. Preprtion of PBCA-TMC nnoprticles Coted nnoprticles were prepred by incubtion of TMC queous solution (2.5 ml, 0.5% w/v) in 5.0 ml of nnoprticles suspension under mgnetic stirring (50 rpm) for 1 h. Method vlidtion Reference stndrd stock solutions Stndrd ACV ws ccurtely weighed (100.0 mg) nd trnsferred to ml volumetric flsk. HCl solution (0.1 M, 1.0 ml) ws dded to promote totl dissolution nd the volume ws completed with MeOH:ACN (8:2 v/v). This solution ws diluted with the sme solvent, obtining the ACV concentrtion of 20.0 µg/ml. Plcebo solutions PBCA nnoprticles (plcebo 1) nd PBCA-TMC nnoprticles (plcebo 2) An liquot of 10.0 ml of the PBCA nnoprticles suspension (or PBCA-TMC nnoprticles suspension) ws trnsferred to ml volumetric flsk followed by ddition of HCl solution (0.1 M, 1.0 ml), nd the volume ws completed with MeOH:ACN (8:2 v/v). Then, 5.0 ml were trnsferred to ml volumetric flsk nd the volume ws completed with the sme solvent. Finlly, 15.0 ml were centrifugted ( rpm, 60 min, t room temperture) nd the superntnt ws used for mesurements. Smple solutions PBCA nnoprticles with ACV (smple 1) nd PBCA-TMC nnoprticles with ACV (smple 2) Aliquots of mg of ACV nd 10.0 ml of PBCA nnoprticles suspension (or PBCA-TMC nnoprticles with ACV) were trnsferred to ml volumetric flsk followed by the ddition of HCl solution (0.1 M, 1.0 ml) to promote totl dissolution of ACV nd the volume ws completed with MeOH:ACN (8:2 v/v). Then, 5.0 ml of this solution were trnsferred to ml volumetric flsk nd the volume ws completed with the sme solvent. The finl ACV concentrtion ws 20.0 µg/ml. Finlly, 15.0 ml of this lst solution ws centrifugted ( rpm, 60 min, t room temperture) nd the superntnt ws used for mesurements. Blnk solution Blnk solution ws prepred by trnsferring 1.0 ml of 01.M HCl solution to ml volumetric flsk nd completing volume with MeOH:ACN (8:2 v/v). Specificity The specificity of the method ws evluted by the nlysis of stndrd solution, plcebo solutions (1 nd 2) s well s smple solutions (1 nd 2). Zero-order spectr were obtined over the nm rnge. From this, first-order derivtive spectr were obtined. Linerity Aliquots from the stock solutions of ACV were trnsferred to volumetric flsks nd diluted with blnk solution to obtin the finl concentrtions of 1.25, 2.5, 5.0, 8.0, 10.0, 16.0, 20.0 nd 40.0 µg/ml. Ech solution ws prepred in triplicte. The first-order derivtive spectr (zero-crossing method) were obtined over the nm rnge nd determintions were mde t nm. The linerity ws evluted by liner regression nlysis, which ws clculted by the lest squre regression method. Precision nd ccurcy Aliquots of 10.0 ml from plcebos 1 nd 2 were dded to known mounts of stndrd solutions of ACV (25.0, nd µg/ml). Ech smple ws diluted with blnk solution to nlyticl concentrtions (2.5, 10.0 nd 20.0 µg/ml), centrifugted ( rpm, 60 min, t room temperture) nd superntnts were used for mesurements. Precision nd ccurcy were clculted intr- nd inter-dy s RSD nd E, respectively, s estblished by ICH in the Vlidtion of Anlyticl Procedure Methodology. 26 Detection (LOD) nd Quntifiction (LOQ) limits Detection nd quntifiction limits were estimted by the slope nd men stndrd devition of stndrd ACV concentrtions employed to construct the clibrtion curve. 26 Recovery Aliquots of smples 1 nd 2 (20.0 µg/ml of ACV) were dded to known mounts of stndrd solutions of ACV (80.0, nd µg/ml). Ech smple ws diluted with blnk solution to nlyticl concentrtions (10.0, 15.0 nd 20.0 µg/ml). Solutions were recorded in the rnge of nm nd determintions were mde t nm. Recovery ws performed in nine replictes. 26

4 Vol. 35, No. 1 Derivtive spectrophotometric method for determintion of cyclovir 205 RESULTS AND DISCUSSION The literture describes the use of different solvents in order to disrupt the PBCA nnoprticles for subsequent quntifiction of the ssocited drug, such s chloroform, 12 ethnol, 13 methnol 16 nd cetonitrile. 27 These solvents, s well s their mixtures in different proportions, were evluted through visul observtion with regrd to their bility of efficiently disrupt nnoprticles. The best solvent composition ws found to be MeOH:ACN (8:2, v/v); however, previous ddition of HCl solution (0.1 M, 1.0 ml) (ph 1.2) ws necessry to ensure complete solubiliztion of ACV. The zero-order spectr from ACV in PBCA or PBCA-TMC nnoprticles showed complete overlpping s cn be observed in Figure 2. Therefore clssicl UV method cnnot be pplied. So, to ssure no significnce of these signls from plcebos, nominl derivtive vlues were evluted. The first-derivtive ( 1 D) spectr from plcebo 1 nd 2, ACV, nd nnoprticle systems (uncoted nd coted with TMC) were presented by Figure 3. In this figure, it is possible to observe tht ll interferences were cncelled out t the wvelength of nm. Figure 2. Zero-order bsorbnce spectr of () PBCA-TMC with ACV (20.0 µg/ml), (b) PBCA with ACV (20.0 µg/ml), (c) ACV (20.0 µg/ml), (d) PBCA- TMC, (e) PBCA. Abs = bsorbnce Figure 3. First-order derivtive spectr of () PBCA with ACV (20.0 µg/ml), (b) ACV (20.0 µg/ml), (c) PBCA-TMC with ACV (20.0 µg/ml), (d) PBCA, (e) PBCA-TMC In ddition, in order to improve the 1 D method, different smoothing nd scling fctors were tested, where smoothing fctor of Δλ = 2.0 nd scling fctor = 1.0 showed suitble signl-to-noise rtio nd spectr showed good resolution. A liner correltion (r = ) ws obtined between pek mplitude t nm nd the corresponding concentrtion for ACV in the rnge of 1.25 nd 40.0 µg/ml (Figure 2S, supplementry mteril). The liner regression eqution ws found to be: y = x Intr- nd inter-dy precision, clculted s RSD, nd ccurcy vlues re reported in Tbles 1 nd 2. Reltive smll mount of RSD, not exceeding the limit of 5.0%, with men vlues for intr- nd interrun ssys were 0.79 nd 1.87% for the PBCA nnoprticles (plcebo 1). For the PBCA-TMC nnoprticles (plcebo 2) these vlues were 0.65 nd 2.89%, respectively, nd confirmed the precision, reproducibility nd repetbility of the first derivtive method t nm. Accurcy men vlues (intr- nd inter-dy) were nd 101.0% for the PBCA nnoprticles (plcebo 1), wheres for the PBCA-TMC nnoprticles (plcebo 2) were nd 100.7%. LOD nd LOQ were estimted s 0.08 µg/ml nd 0.25 µg/ml, respectively. Recovery experiment ws conducted to corroborte the ccurcy of the proposed zero-crossing first-derivtive spectrophotometric method. The men recovery vlue (n = 9) for PBCA nd PBCA-TMC smples with stndrd ACV ws nd 100.2, respectively (Tbles 3 nd 4), indicting cceptble ccurcy. CONCLUSIONS Up to now, no derivtive procedures hve been described for ssy of ACV present in PACA nnoprticles preprtions. As result, new derivtive spectrophotometric method ws developed for determintion of this drug in polymeric nnoprticles coted or uncoted with TMC. This described method hs eliminted the interference from excipients, llowing the quntifiction of ACV in these formultions with good ccurcy. The method showed to be simple, rpid, sensitive Tble 1. Intr- nd inter-dy ssys for determintion of precision nd ccurcy of DS method (295.2 nm, zero-crossing) for plcebo 1 Intr-dy Inter-dy Anlyticl concentrtions (µg/ml) Mens ± s.d. (µg/ml) RSD (%) E (%) Mens b ± s.d. (µg/ml) RSD (%) E (%) ± ± ± ± ± ± n = 3; b n = 9; s.d. = stndrd devition; precision = reltive stndrd devition (RSD); E = ccurcy. Tble 2. Intr- nd inter-dy ssys for determintion of precision nd ccurcy of DS method (295.2 nm, zero-crossing) for plcebo 2 Intr-dy Inter-dy Anlyticl concentrtions (µg/ml) Mens ± s.d. (µg/ml) RSD (%) E(%) Mens b ± s.d. (µg/ml) RSD (%) E (%) ± ± ± ± ± ± n = 3; b n = 9; s.d. = stndrd devition; precision = reltive stndrd devition (RSD); E = ccurcy.

5 206 Tvres et l. Quim. Nov Tble 3. Results obtined from recovery tests of ACV from PBCA nnoprticles by DS method (295.2 nm, zero-crossing) Concentrtion (µg/ml) Stndrd Smple Finl concentrtion Found (men ± s.d.) Recovery (%) ± ± ± n = 3; s.d. = stndrd devition. Tble 4. Results obtined from recovery tests of ACV from PBCA-TMC nnoprticles by DS method (295.2 nm, zero-crossing) Concentrtion (µg/ml) Stndrd Smple Finl concentrtion Found (men ± s.d.) Recovery (%) ± ± ± n = 3; s.d. = stndrd devition. nd precise nd could be esily pplied in the determintion of ACV ssocited to these nnocrries. Hence, first-derivtive presented to be n useful nlyticl method for precise nd ccurte determintion of ACV in PBCA nd PBCA-TMC nnoprticles. SUPPLEMENTARY MATERIAL Avilble t in pdf file, with free ccess. ACKNOWLEDGMENTS The uthors would like to thnk CNPq nd CAPES for scholrships finncil support, nd lso Aché Phrmceuticls Lbortories nd Medley Phrmceuticl Industry for gently supply cyclovir phrmceuticl grde. REFERENCES 1. Mohnrj, V. J.; Chen, Y.; Trop. J. Phrm. Res. 2006, 5, Mllipeddi, R.; Rohn, L. C.; Int. J. Nnomedicine 2010, 5, Ishihr, T.; Mizushim, T.; Expert Opin. Drug Deliv. 2010, 7, Suri, S. S.; Fenniri, H.; Singh, B.; J. Occup. Med. Toxicol. 2007, 2, Vuthier, C.; Dubernet, C.; Fttl, E.; Pinto-lphndry, H.; Couvreur, P.; Adv. Drug Deliver. Rev. 2003, 55, Chuvierre, C.; Leclerc, L.; Lbrre, D.; Appel, M.; Mrden, M. C.; Couvreur, P.; Vuthier, C.; Int. J. Phrm. 2007, 338, Brvo-osun, I.; Vuthier, C.; Chcun, H.; Ponchel, G.; Eur. J. Phrm. Biophrm. 2008, 69, Jonker, C.; Hmmn, J. H.; Kotzé, A. F.; Int. J. Phrm. 2002, 238, Moury, V. K.; Inmdr, N. N.; J. Mter. Sci.: Mter. Med. 2009, 20, Werle, M.; Tkeuchi, H.; Bernkop-Schnürch, A.; J. Phrm. Sci. 2009, 98, Polnok, A.; Borchrd, G.; Verhoef, J. C.; Srisut, N.; Junginger, H. E.; Eur. J. Phrm. Biophrm. 2004, 57, Weiss, K.; Lorenz, M.; Lndfester, K.; Milnder, V.; Mcromol. Biosci. 2007, 7, Mulik, R.; Mhdik, K.; Prdkr, A.; Eur. J. Phrm. Sci. 2009, 37, Liu, H.; Chen, J.; PDA J. Phrm. Sci. Tech. 2009, 63, Xi-xio, Y.; Jn-hi, C.; Shi-ting, L.; Dn, G.; Xv-xin, Z.; Reg. Tox. Phrm. 2006, 46, He, M.; Zho, Z.; Yin, L.; Tng, C.; Yin, C.; Int. J. Phrm. 2009, 373, Luo, D. Q.; Guo, J. H.; Wng, F. J.; Jin, Z. X.; Cheng, X. L.; Zhu, J. C.; Peng, C. Q.; Zhng, C.; J. Biomter. Sci., Polym. Ed. 2009, 20, Siqueir-Mour, M. P.; Lir, M. C. B.; Sntos-Mglhães, N. S.; Rev. Brs. Cienc. Frm. 2008, 44, Krpinsk, J.; Tlnt 2004, 64, Bosch Ojed, C.; Snches Rojs, F.; Anl. Chim. Act 2004, 518, Mendez, A. S. L.; Deconto, L.; Grci, C. V.; Quim. Nov 2010, 33, Mhrous, M. S.; Abdel-Khlek, M. M.; Dbees, H. G.; Beltgy, Y. A.; Anl. Lett. 1992, 25, Dbees, H. G.; Anl. Lett. 1998, 31, Curti, E.; Britto, D.; Cmpn-Filho, S. P.; Mcromol. Biosci. 2003, 3, Couvreur, P.; Vuthier, C.; J. Controlled Relese 1991, 17, ICH - Interntionl Conference on Hrmoniztion of Technicl Requeriments for Registrtion of Phrmceuticls for Humn Use, Q2B(R1): Guideline on Vlidtion of Anlyticl Procedure Methodology, Mksimenko, O.; Pvlov, E.; Toushov, E.; Molin, A.; Stuklov, Y.; Prudskov, T.; Feldmn, V.; Kreuter, J.; Gelperin, S.; Int. J. Phrm. 2008, 356, 325.

6 Quim. Nov, Vol. 35, No. 1, S1-S2, 2012 DERIVATIVE SPECTROPHOTOMETRIC METHOD FOR DETERMINATION OF ACYCLOVIR IN POLYMERIC NANOPARTICLES Guilherme Diniz Tvres*, Gisele Miki Ishikw, Tlit Ferreir Monteiro, Crolin Znolini, Érik Ros Mri Kedor- Hckmnn, Nádi Arci Bou-Chcr e Vldi Olg Consiglieri Fculdde de Ciêncis Frmcêutics, Universidde de São Pulo, Av. Prof. Lineu Prestes, 580, São Pulo - SP, Brsil Supplementry Mteril Figure 1S. 1 H-NMR spectr of the TMC *e-mil: diniztvres@usp.br

7 S2 Tvres et l. Quim. Nov Figure 2S. First-order derivtive overlpped spectr of ACV stndrd. Concentrtions were 1.25, 2.5, 5.0, 8.0, 10.0, 16.0, 20.0 nd 40.0 µg/ml