PRATIK KUMAR GUPTA*, VIBHA CHATURVEDI. Department of Chemistry, Mewar University, Chittorgarh, Rajasthan, India.

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1 Vol 10, Issue 11, 2017 Online Print Reserch Article A STUDY ON FORCED DEGRADATION AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR DETERMINATION OF BENZALKONIUM CHLORIDE IN AZELASTINE HYDROCHORIDE PHARMACEUTICAL OPHTHALMIC FORMULATION ABSTRACT PRATIK KUMAR GUPTA*, VIBHA CHATURVEDI Deprtment of Chemistry, Mewr University, Chittorgrh, Rjsthn, Indi. Emil: pkpipriy@gmil.com Received: 30 July 2017, Revised nd Accepted: 29 August 2017 Ojective: This study ws designed to conduct forced degrdtion nd vlidtion studies for determintion the enzlkonium chloride (BKC) s mjor homologues form in zelstine hydrochloride phrmceuticl ophthlmic formultion y novel stility indicting reverse phse high-performnce liquid chromtogrphic method (RP-HPLC). Methods: Forced degrdtion study nd vlidtion were crried out with nlyticl pproch of reverse phse chromtogrphic nlysis with C18, cosmosil (250mm x 4.6mm i.d. x 5µm) column using moile phse consisting cetonitrile uffer of ph 5.0 with 5N NOH (45:55 %v/v) t flow rte of 1.5 ml per minute. Column temperture ws mintined t 25 C nd detection wvelength ws 210nm.By using these chromtogrphic conditions of method, two mjor homologues of enzlkonium chloride were seprted without ny interference of drug components nd exciepients. Results: Forced degrdtion studies were crried out s per ICH guidelines nd estlished stility indicting property of method.the method ws found liner in concentrtion rnge 40µg/ml to 60µg/ml (50µg/ml±20%), correltion coefficient ws found The recovery ws found to e 99.2%, 100.7% nd 101.4% t ±30% of trget concentrtion. The %RSD for method precision nd intermedite precision were found 0.28% nd 0.30 % respectively. The method ws found specific, selective, precise, ccurte, liner, roust nd stility indicting for quntifiction enzlkonium chloride in zelstine phrmceuticl ophthlmic formultion. Conclusion: The method hs een found successful for determintion of BKC phrmceuticl formultion nd vlidted for ll the prmeters of vlidtion nd found within the cceptnce criteri s per ICH guideline Q2R1. Keywords: Benzlkonium chloride content, HPLC, BKC, Preservtive, ophthlmic solution, forced degrdtion The Authors. Pulished y Innovre Acdemic Sciences Pvt Ltd. This is n open ccess rticle under the CC BY license ( org/licenses/y/4. 0/) DOI: INTRODUCTION Benzlkonium chloride (BKC) is preservtive most commonly found in ophthlmic nd nsl formultions [1]. BKC is quternry mmonium compound, mixture of lkylenzyldimethylmmonium chlorides contining the formul [C6H5CH2N(CH3)2R]Cl, where R is n lkyl group vrying from C8H17 to C18H37 (Fig. 1) [2]. As n ntimicroil gent, BKC cn e sfely used t concentrtions from 0.002% to 0.02% ut it cn e up to 0.2% depending on vrious fctors in ophthlmic drug products [3]. BKC is common preservtive used in ophthlmic drug products with the concentrtions rnge from 0.004% to 0.01%. Higher concentrtions cn e custic [4] nd it my cuse irreversile dmge to the cornel endothelium. Vrious reserches on BKC hve found no direct reltion etween the use of BKC in concentrtions elow 0.1% in nsl sprys nd drug-induced rhinitis [5]. BKC solutions ct s iocidl gents with firly long durtion of ction. They re strongly ctive ginst some viruses, cteri, fungi, nd protozo. Bcteril spores re considered to e more resistnt. These BKC solutions re cteriosttic or ctericidl ccording to their concentrtion. Sensitivity of Grm-positive cteri is more thn other. Azelstine hydrochloride phrmceuticl ophthlmic formultion is reltively selective H1 receptor ntgonist for topicl dministrtion to eyes. Azelstine hydrochloride hs ntihistminic therpeutic effects vulnerle providing immedite relief, mst cell stiliztion providing erly-phse intervention, nd inhiition of expression nd ctivtion of nti-inflmmtory meditors which chrcterize the lte phse of the immune rection [6]. Its chemicl nme is (±)-1-(2H)-phthlzinone,4- [(4chlorophenyl) methyl]-2-(hexhydro-1-methyl-1h-zepin-4-yl)-, monohydrochloride. Its moleculr formul is C 22 H 24 ClN 3 O HCl with the following chemicl structure (Fig. 2). A numer of nlyticl methods hve een reported for the estimtion of BKC in vrious products including ophthlmic preprtions [7-23]. However, there ws no method ville for estimtion of BKC in zelstine hydrochloride ophthlmic formultions. Severl chromtogrphic methods hve een developed for the determintion of BKC in the pst. The quntifiction of BKC using these methods in ophthlmic solutions ws not suitle for our product of interest. Low detection sensitivity ecuse of high rnge of wvelength utiliztion, long run-time nd use of reversed-phse CN column is criticl with some types of ophthlmic drug products. As our ophthlmic solution contined zelstine s ctive ingredient nd other excipients, injection t high concentrtion ws not suggested with the CN column. The idel method should hve high sensitivity of detector, minimum smple concetrtionion to mintin column roustness, nd optimized low run-time nlysis. Dudkiewicz-Wilczynsk et l. reported used phenomenex Lun CN, 250 mm 4.6 mm 5 µm column nd 0.075M cette uffer, ph5.0 with cetonitrile in rtio of (55:45 v/v) nd flow rte ws 1.5 ml/minute. The detecton wvelength is 262 nm nd column temperture is 25 C [22].

2 conducted for optimiztion of smple concentrtion nd stndrd solution concentrtion. Buffer ws prepred y dissolving 5.0 ml of orthophosphoric cid in 1000 ml of wter nd dded 1.0 ml of triethyl mine to this solution. The ph of uffer ws djusted to 5.0 with 5 N NOH solutions. The moile phse consisted of uffer ph 5.0: Acetonitrile (55: 45, v/v) ws filtered y 0.45 µ memrne filter nd degssed under vcuum efore use. The flow rte of pump for moile phse ws 1.5 ml/minute. The UV detection ws performd t 210 nm nd the injection volume ws µl. The HPLC system equipped with mintining column oven temperture with 25 C. The reverse phse column cosmosil C18, 250 mm length 4.6 mm I.D. 5 µm prticle size ws used for optimiztion of chromtogrphic conditions. Fig. 1: Chemicl structure of enzlkonium chloride Stndrd nd smple solution preprtion Stndrd preprtion A working stndrd solution of BKC (50 µg/ml) ws prepred using diluent for ssy determintion. Smple preprtion Ophthlmic solution contining BKC (50 µg/ml) ws prepred using diluent. System suitility prmeters Five replictes injections of stndrd, nd the system suitility check ws doe s follows. 1. The percentge reltive stndrd devition (RSD) of the totl re of homologues peks should e not more thn The theoreticl pltes of oth peks should e not < The tiling fctor of oth peks should e not more thn 2.5. Louis-Philppe et l. reported tht column wters symmetry shield RP18 (75 mm 4.6 mm 3.5 µm) ws chosen. The moile phse ws rtio (68:32 v/v) of methnol-potssium phosphte (ph3.0, 7.5 mm). The flow rte ws 1.0 ml/minute t column oven temperture 50 C. The wvelength ws 208 nm nd injection volumn ws 20 µl [23]. The min ojective of this work ws the determintion of BKC in zelstine hydrochloride ophthlmic solution y reverse phse high-performnce liquid chromtogrphic (HPLC) method development, vlidtion, nd roustness. METHODS Fig. 2: Chemicl structure of zelstine hydrochloride Regent nd chemicls The stndrd of BKC 50% solution with 50% purity ws provided y fisher scientific, Mumi. HPLC grde cetonitrile, wter, nd HCl (AR grde) were otined from Merck, Mumi. H 2 O 2, sodium hydroxide, orthophsphoric cid were otined from fisher scientific, Mumi. All regents were used without ny further purifiction. All components were otined from commercil sources nd used s received. Instrumenttion Anlysis ws performed on n wters 2695 HPLC system nd Shimdzu HPLC system equipped ultrviolet (UV) nd photo diode rry detector equipped quternry solvent delivery pump, utomtic smple injector, column thermostt. The dt cquisition nd nlysis were performed using Empower softwre nd LC solution. Method nd conditions The HPLC equipped UV/photo diode rry detector ws used s nlyticl technique for the determintion of BKC homologs in zelstine ophthlmic solution. Detector response versus smple preprtion ws optimized in series of experiments where other prmeters such s column selection, moile phse, column temperture, nd different diluents were nlyzed. In ddition, some set of experiments hve een Method vlidtion The method vlidtion [24,25] for BKC determintion ws performed s per ICH guideline y determining the prmeters: Specificity, precision, linerity, ccurcy or recovery, roustness, nd forced degrdtion studies. Specificity Specificity is the ility to ssess unequivoclly the nlyte in the presence of components which my e expected to e present. The specificity of method ws determined y nlyzing the solutions such s diluent, stndrd solution of BKC, nd ophthlmic smple solution of 100% level. Ech of the solution is injected on HPLC equipped with photo diode rry detector for pek purity nlysis nd chromtogrms were recorded. Instrument precision System precision ws estlished y injecting five replicte injections of stndrd solution of BKC (50 µg/ml). The pek res of sum of homolog s peks, tiling fctor, nd theoreticl pltes of ech homolog s were evluted. Method precision The precision of n nlyticl procedure expresses the closeness of greement (degree of sctter) etween series of mesurements otined from multiple smpling of the sme homogeneous smple under the prescried conditions. Method precision ws determined y nlysis of BKC in six ophthlmic solution preprtions nd ech smple ws injected in duplicte. For ech smple, chromtogrms were recorded. The percentge RSD of six ssy of smple preprtion ws clculted nd found <2.0. Intermedite precision Intermedite precision expresses within - lortories vritions: Different dys, different nlysts, different equipment, etc. The min ojective of intermedite precision is to find out the effect of rndom events on the nlyticl method so tht nlysis of smple on different column, different dy nd different instrument. 375

3 Accurcy The ccurcy of n nlyticl procedure expresses the closeness of greement etween the vlue which is ccepted either s conventionl true vlue or n ccepted reference vlue nd the vlue found. Accurcy of method is defined s the closeness of mesured vlue to the true vlue for smple. Accurcy of HPLC method ws crried out three different level, i.e. ±30% of level clim, so tht, 70%, 100%, nd 130% of BKC. Three smples were prepred t ech concentrtion level nd injected in duplicte. Linerity/rnge The linerity of n nlyticl procedure is its ility (within given rnge) to otin test results which re directly proportionl to the concentrtion (mount) of nlyte in the smple. The rnge of n nlyticl procedure is the intervl etween the upper nd lower concentrtion (mounts) of nlyte in the smple (including these concentrtions) for which it hs een demonstrted tht the nlyticl procedure hs suitle level of precision, ccurcy, nd linerity. Linerity ws performed to verify detector limit in minimum of five concentrtion level etween ±20% of 100% level. A minimum five concentrtion level of stndrd solution ws injected in duplicte. As outcome of linerity slope, Y- inetcept nd correltion coefficient hve een clculted. Roustness The roustness of n nlyticl procedure is mesure of its cpcity to remin unffected y smll, ut delierte vritions in method prmeters nd provides n indiction of its reliility during norml usge. Deliverle chnges in method prmeters The study hs een conducted for effect of smll vrition in method prmeters for optimiztion of method such s: 1. Column oven temperture vrition (±2 C) 2. Vrition in flow rte (±10%) 3. Vrition in ph of uffer (±0.2 unit) 4. Vrition of moile phse composition (±1%). The stndrd solution of BKC nd smple solution were injected for ech of the roustness conditions (such s column temperture ±2 C, ph of uffer ±0.2 unit nd moile phse composition ±1%). Forced degrdtion studies For proving stility indicting strength of method, forced degrdtion studies hve een conducted [26,27]. Stility indicting method mens tht our method is cple of ccurte nd precise mesurement of ctive ingredients (drug sustnce n drug products) free from process impurities, excipients, nd degrdtion products. Acid degrdtion 5 ml of smple solution ws trnsferred into volumetric flsk of 20 ml. Added 2 ml of 1M HCl to this solution nd flsk ws kept this solution t 80 C for 2 hrs in wter th. After 2 hrs, smple withdrwn nd llow to come t room temperture, neutrlized y 1 M NOH solution nd diluted to finl concentrtion of 50 µg/ml, nd injected immeditely. Therml degrdtion 5 ml of smple solution ws trnsferred into volumetric flsk of 20 ml. Flsk ws kept t 80 C for 2 hrs in wter th. After 2 hrs, smple withdrwn nd diluted to finl concentrtion of 50 µg/ml nd injected. RESULTS AND DISCUSSION Method development nd optimiztion of chromtogrphic conditions Primry ojective of this study is determintion of BKC content in zelstine hydrochloride ophthlmic solution. As per literture serch, previously vrious chromtogrphic methods for BKC determintion hve een reported. Different column hve een used such s reverse phse columns (C8 or C18), CN column. Vrious columns such s L10 or CN pcking, L1(C18) were used for determintion of BKC y HPLC. Moile phse consisting of queous uffer ph were used with cetonitrile or methnol in different rtio [27]. As per effects of vrious fctors were nlyzed for seprtion process for determintion of conditions of BKC homologues. The influence of vrious column types, moile phse rtio nd column oven temperture, flow rte, nd different wvelength were summrized. After optimiztion of vrious column nd uffer with different ph rnge with moile phse consisting of cetonitrile nd uffer, finlly, we hve tken trils on cosmosil column 250 mm 4.6 mm 5 µm using moile phse comprising of cetonitrile: Buffer. Vrious tril hve een tken for optimizing the flow rte, moile phse composition, column temperture, wvelength selection, nd finlized the chromtogrphic condition sed on optimiztion results. Optimiztion of smple preprtion Optimiztion of smple preprtion ws chllenging tsk due to complex composition of ophthlmic formultion. Aqueous diluting solution of smple preprtion creted foming nd orgnic mixed diluents creted the precipittion of some exciepients. Vrious diluents were tested, the moile phse ws found to e the est diluents for getting good recovery of totl BKC s per ICH guideline criteri. Using this type of smple preprtion, recovery results otined etween 98% nd 102%. Typicl chromtogrms were otined for moile phse, stndrd solution, nd smple solution contining zelstine ctive drug re presented in Figure, s shown in Figs Two peks of BKC were found in chromtogrms otined from oth stndrd solution nd smple solution. Method vlidtion System suitility The system suitility ws performed to for verify the chromtogrph t the time of opertion y evlution of chromtogrphic prmeters from six replicte injection of stndrd solution of BKC (50 µg/ml in diluent). The results is clculted for ech homolog pek of BKC nd found cceptle s shown in Tle 1. Specificity Specificity of nlyticl method is its ility to mesure ccurtely nd specificlly the nlyte of interest in the presence of smple Alkli degrdtion 5 ml of smple solution ws trnsferred into volumetric flsk of 20 ml. Added 2 ml of 1M NOH to this solution nd flsk ws kept this solution t 80 C for 2 hrs in wter th. After 2 hrs, smple withdrwn nd llow to come t room temperture, neutrlized y 1 M HCl solution nd diluted to finl concentrtion of 50 µg/ml, nd injected immeditely. Oxidtive degrdtion 5 ml of smple solution ws trnsferred into volumetric flsk of 20 ml. Added 2 ml of H 2 O 2 to this solution nd flsk ws kept t 80 C for 2 hrs in wter th. After 2 hrs, smple withdrwn nd diluted to finl concentrtion of 50 µg/ml, nd injected. Fig. 3: Representtive chromtogrm for moile phse 376

4 mtrix, in this study, the ility of the methods to seprte the drug from its degrdtion products without the interference of other smple components indicted the specificity of the developed method. Specificity/selectivity of method ws determined y nlysis diluents, stndrd solution, nd smple solution. No significnce interference ws oserved t the retention time of homolog peks of BKC, so tht, it is proved tht method ws specific for nlysis of BKC in zelstine hydrochloride ophthlmic solution s shown in Figs In wters, HPLC system pek purity ngle should e less thn pek purity threshold. In interprettion of chromtogrms, for oth peks of BKC, the pek purity ngle ws less thn pek purity threshold. So tht, this purity fctors declres tht there is no coelution of other peks s shown in Figs. 6 nd 7. Linerity The linerity of method revels the liner reltionship of response ginst the selected concentrtion of nlyte. Linerity of method ws estlished s liner regression nlysis with lest squre method on stndrd solution of BKC. Five stndrd solutions contining BKC comprises 80%, 90%, 100%, 110%, nd 120% of 100% trget level corresponding to µg/ml. The clirtion curve ws constructed y plotting pek re versus concentrtion of enzlkonium chloride. Regression line ws estlished y lest squres method. A correltion coefficient of ws otined s shown in Fig. 8 nd results re discussed in Tle 2. Precision The method precision ws performed from six smple prepred nd injected in duplicte nd percentge RSD ws otined within cceptle limit 2. Hence, the method is reproducile. The low percentge RSD indictes tht the method is precise nd reproducile for quntifiction of BKC content s preservtive in this ophthlmic solution. Intermedite precision ws performed for y injecting six smple preprtions on different dy, y different column, nd different instrument. Percentge ssy is clculted for six determintions nd percentge RSD ws found 0.30% for intermedite precision s shown in Tle 3. There ws no significnce difference found in intermedite conditions of nlysis. So tht, we cn sy tht method is precise for intended purpose in different lortories. Accurcy/recovery Method ccurcy ws evluted y prepring the smple solution t 70%, 100%, nd 130% level of totl enzlkoium chloride leled clim, i.e µg/ml, µg/ml, nd µg/ml rrecovery clculted for ech level re 99.2%, 100.7% nd 101.4%.The %RSD ws clculted 0.74%, 1.21% nd 0.47 respectively nd overll RSD ws clculted 0.81% s shown Tle 4.The results of recovery studies indicted tht the method is ccurte for estimtion of BKC in zelstine ophthlmic formultions. Fig. 4: Representtive chromtogrm for stndrd solution Tle 1: Evlution of system suitility prmeters for stndrd solution Prmeters Acceptnce criteri BKC pek 1 BKC pek 2 Retention time %RSD Pek re %RSD Tiling fctor < Theoreticl pltes > Resolution > RSD: Reltive stndrd devition, BKC: Benzlkonium chloride Tle 2: Linerity prtmeter for BKC Fig. 5: Representtive chromtogrm for smple solution Prmeter Result Correltion coefficient Slope Y intercept BKC: Benzlkonium chloride Tle 3: Method precision Fig. 6: Pek purity for enzlkonium chloride min pek (wters high-performnce liquid chromtogrphic) S. No. Method precision Intermedite precision Men RSD % difference 0.8 RSD: Reltive stndrd devition 377

5 Roustness Roustness of proposed method demonstrted non-significnt ltertion through the nlysis of smple solution nd stndrd solution. The results otined in roustness conditions were compred Fig. 7: Pek purity for enzlkonium chloride minor homologue (Wters high-performnce liquid chromtogrphic) with tht of optimized method. Roustness nlysis of BKC ws crried out with some delierte chnges in chromtogrphic prmeters such s wvelength, column temperture, flow rte, chnges in moile phse rtio, nd ph of moile phse, s representtive chromtogrms s shown in Figs The system suitility prmeters such s theoreticl pltes, percentge RSD, nd tiling fctor were found within the limit s shown Tle 5. Forced degrdtion studies BKC content ws determined in different stress condition of forced degrdtion study. No significnce interference ws found t the retention time of BKC peks. There ws no interference with zelstine peks t the retention time of BKC peks. The entire peks were well seprted nd cn e esily quntified. The pek purity ngle ws found greter thn pek purity threshold so tht pek ws found pure nd homogenous in every stress conditions, due to which, method confirms its specificity nd stility indicting property [20,28,29]. In lkli degrdtion, 5.4% degrdtion chieved. This method ws found stility indicting method. Representtive chromtogrms re shown in Figs nd results re tulted in Tle 6. Fig. 8: Linerity curve for enzlkonium chloride Fig. 9: Representtive chromtogrm of () stndrd control () test control Accurcy level (%) Theoreticl concentrtion (ctul mount dded in µg/ml) Tle 4: Method ccurcy for BKC determintion Experimentl concentrtion Actul mount recovered in (µg/ml) Recovery (%) Averge (%) % RSD Overll RSD RSD: Reltive stndrd devition, BKC: Benzlkonium chloride 378

6 Tle 5: Results of roustness conditions HPLC condition Tiling fctor Theoriticl pltes %RSD Pek 1 Pek 2 Pek 1 Pek 2 Pek 1 Pek 2 Controlled condition Column temperture minus Column temperture plus Flow rte minus 1.35 ml/minute Flow rte plus 1.65 ml/minute PH of uffer ph of uffer Orgnic plus Orgnic minus Tle 6: Results of forced degrdtion study Stress condition BKC ssy % degrdtion Pek purity (Pek 1) Pek purity (Pek 2) Control condition 99.4 Psses Psses Acid degrdtion Psses Psses Alkli degrdtion Psses Psses Oxidtive degrdtion Psses Psses Therml degrdtion Psses Psses Fig. 10: Representtive chromtogrm of () temperture minus, () temperture plus Fig. 11: Representtive chromtogrm of () flow minus, () flow plus Fig. 12: Representtive chromtogrm () ph minus, () ph plus 379

7 Fig. 13: Representtive chromtogrm of () orgnic minus, () orgnic plus Fig. 14: Representtive chromtogrm for control smple Fig. 15: Representtive chromtogrm for cid degrdtion smple Fig. 16: Representtive chromtogrm for lkli degrdtion smple 380

8 Fig. 17: Representtive chromtogrm for peroxide degrdtion Fig. 18: Representtive chromtogrm for therml degrdtion CONCLUSION This study presents simple, reverse phse-stility indicting HPLC method for determintion of enzlkonium chloride s mjor homologs in zelstine hydrochride phrmceuticl ophthlmic formultion. The developed nd vlidted method is specific, selective, precise, liner, ccurte, nd roust. All the degrdtion products were well seprted from nlyte peks demonstrting tht the developed method ws specific nd stility indicting. This method stility indicting ecuse of its successful evlution of forced degrdtion studies. The vlidtion study indictes tht this method cn e considerle for quntifiction of BKC in zelstine hydrochloride ophthlmic formultion. REFERENCES 1. Budouin C, Lé A, Ling H, Puly A, Brignole-Budouin F. Preservtives in eyedrops: The good, the d nd the ugly. Prog Retin Eye Res 2010;29(4): United Sttes Phrmcopei. USP 40-NF35. Benzlkonium Chloride; p Liu J, Lu GW, Sndovl M, Ciringh Y, Xue G, Jeger D, et l. Determintion of enzlkonium chloride prtition in micelle solutions using ultrfiltrtion method. AAPS PhrmSciTech 2009;10(4): Nelson L, Goldfrnk L. Goldfrnk s Toxicologic Emergencies. 9 th ed. New York: McGrw-Hill Medicl; Mrple B, Rolnd P, Benninger M. Sfety review of enzlkonium chloride used s preservtive in intrnsl solutions: An overview of conflicting dt nd opinions. Off J Am Acd Otolryngol Hed Neck Surg 2004;130: Azelstine Hydrochloride Ophthlmic Solution. 0.05%, OPTIVAR ; Aville from: lel/2000/21127ll.pdf. 7. Agrwl A, Tiwri S, Ngriy K. Method development nd its vlidtion for quntittive simultneous determintion of ltnoprost, timolol nd enzlkonium chloride in ophthlmic solution y RPHPLC. J Drug Deliv Ther 2013;3(2): Al-Fkhory AA, Al-Klk I, Al-Khti MJ. Chromtogrphic determintion of totl enzlkonium chloride [BAC] in some of ophthlmic preprtions y HPLC. Dmscus Univ J Bsic Appl Sci 2014;30: Chipett SC, de Oliveir EC, Olivier BC, Mercnte LA, Henriques DM, Pereir Netto AD. Intrlortory vlidtion, comprison nd ppliction of HPLC-UV-DAD methods for simultneous determintion of enzlkonium chloride, chlorexidine digluconte nd triclosn. J Brz Chem Soc 2011;22(10): Murnne D, Mrtin G, Mrriot C. Vlidtion of reverse phse HPLC method for concurrent ssy of wek se nd phrmcologiclly ctive steroid. J Phrm Biomed Anl 2006;40: Ger M, Shwish HM, Khedr AM, Aed-Almonem KI. Determintion of enzlkonium chloride preservtive in phrmceuticl formultion of eye nd er drops using new potentiometric sensors. Mter Sci Eng C 2012;32(8): Jovovic M, Kostic N, Jncic-Stojnovic B, Mlenovic A. Investigtion of tropicmide nd enzlkonium chloride stility using liquid chromtogrphy. J Liq Chromtogr Relt Technol 2012;32: Kpkyl SV, Mueen G, Kimhune R. Reverse phse-high performnce liquid chromtogrphic method for quntittion of epinstine hydrochloride in eye drops. Indo Am J Phrm Res 2013;3: Kostic DA, Mitic SS, Nskovic DC, Zruic AR, Mitic MN. Determintion of enzlkonium chloride in nsl drops y highperformnce liquid chromtogrphy. J Chem 2012;9(3): Meht J, Ptidr K, Vys N. Development nd vlidtion of precise method for determintion of enzlkonium chloride (BKC) preservtive, in phrmceuticl formultion of ltnoprost eye drops. J Chem 2010;7: Ro PV, Venktesh P, Kumr MR. Vlidted stility indicting UPLC method for the estimtion of enzlkonium chloride in ketorolc tromethmine ophthlmic solution. Int J Chrom Sci 2013;3: Sntos M, Li M, Rustum AM. A single RP-LC method for the determintion of enzlkonium chloride nd its potentil impurities in enzlkonium chloride rw mteril. Chromtogrphi 2010;71(5-6): Shikh KA, Ptil AT. Stility-indicting HPLC method for the determintion of mometsone furote, oxymetzoline, phenyl ethnol nd enzlkonium chloride in nsl spry solution. J Trce Anl Food Drugs 2013;1: Shen Y, Xu SJ, Wng SC, Tu JS. Determintion of enzlkonium chloride in viscous ophthlmic drops of zithromycin y high-performnce 381

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