Anodic Oxidation of Etodolac and its Linear Sweep, Square Wave and Differential Pulse Voltammetric Determination in Pharmaceuticals

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1 Reserch Pper Anodic Oxidtion of Etodolc nd its Liner Sweep, Squre Wve nd Differentil Pulse Voltmmetric Determintion in Phrmceuticls B. YILMAZ*, S. KABAN AND B. K. AKCAY Deprtment of Anlyticl Chemistry, Fculty of Phrmcy, Atturk University, Erzurum, Turkey Yılmz, et l.: Anodic Oxidtion of Etodolc nd Voltmmetric Determintion In this study, simple, fst nd relible cyclic voltmmetry, liner sweep voltmmetry, squre wve voltmmetry nd differentil pulse voltmmetry methods were developed nd vlidted for determintion of etodolc in phrmceuticl preprtions. The proposed methods were bsed on electrochemicl oxidtion of etodolc t pltinum electrode in cetonitrile solution contining 0.1 M lithium perchlorte. The well defined oxidtion pek ws observed t 1.03 V. The clibrtion curves were liner for etodolc t the concentrtion rnge of µg/ml for liner sweep, squre wve nd differentil pulse voltmmetry methods, respectively. Intr nd inter dy precision vlues for etodolc were less thn 4.69, nd ccurcy (reltive error) ws better thn 2.00%. The men recovery of etodolc ws 100.6% for phrmceuticl preprtions. No interference ws found from three tblet excipients t the selected ssy conditions. Developed methods in this study re ccurte, precise nd cn be esily pplied to Etol, Tdolk nd Etodin tblets s phrmceuticl preprtion. Key words: Etodolc, cyclic voltmmetry, liner sweep voltmmetry, squre wve voltmmetry, differentil pulse voltmmetry Nonsteroidl ntiinflmmtory drugs (NSAIDs) re widely used for the tretment of severl rheumtic nd inflmmtory diseses [1 3]. Etodolc (fig. 1), which is 2 [(1RS) 1,8 diethyl 1,3,4,9 tetrhydropyrno[3,4 b] indol 1 yl] cetic cid [4], is chirl NSAID with single stereogenic crbon tom ndexists in two enntiomeric forms; R nd S. It is mrketed s rcemte nd used s nlgesic nd for the tretment of the signs nd symptoms of rheumtoid rthritis nd osteorthritis [5]. Etodolc decreses the synthesis of peripherl prostglndins involved in mediting inflmmtion by inhibition of the cycloxygense (COX) enzyme [5]. The drug is selective inhibitor for COX 2 enzyme nd it hs been reported tht its selectivity towrds COX 2 enzyme is 10 times tht of COX 1 enzyme [6]. It produces less gstrointestinl toxicity compred to the other NSAIDs. The rcemte drug is rpidly bsorbed fter orl dministrtion nd is highly bound to plsm protein (99.3%) [7]. It is metbolized in the liver to inctive metbolites *Address for correspondence E mil: yilmzb@tuni.edu.tr tht re primrily eliminted vi the renl route. Etodolc is dministered orlly for ptients with rheumtoid rthritis t doses of mg given twice dily in the form of tblets or cpsules. The drug enntiomers possess virtully identicl physicl nd chemicl properties [8]. On the other hnd, the phrmcologicl properties of the two enntiomers differ profoundly in which the S form is responsible for the nti inflmmtory ctivity [8]. Most of the chirl NSAIDs minly undergo in vivo unidirectionl inversion mechnism; medited by heptic enzymes; from the inctive R form to the ctive S enntiomer [9]. Etodolc does not show in vivo inversion of its enntiomers [10]. This is n open ccess rticle distributed under the terms of the Cretive Commons Attribution NonCommercil ShreAlike 3.0 License, which llows others to remix, twek, nd build upon the work non commercilly, s long s the uthor is credited nd the new cretions re licensed under the identicl terms. For reprints contct: reprints@medknow.com Accepted 26 July 2015 Revised 22 Jnury 2015 Received 16 July 2014 Indin J Phrm Sci 2015;77(4): Indin Journl of Phrmceuticl Sciences Published by Wolters Kluwer - Medknow 413

2 Fig. 1: Chemicl structure of etodolc. Since enntiomers of rcemte drugs exhibit pronounced phrmcokinetic differences, resulting from stereoselective phrmcokinetic processes such s bsorption, distribution, metbolism nd excretion, it should be noted tht the degree of enntioselectivity in phrmcokinetics is mrkedly species dependent nd the dt cnnot be trnsposed between species [11]. In view of these differences, studies of the phrmcokinetics of chirl NSAIDs should lwys mesure nd quntify the seprte enntiomers nd not totl drug. The totl drug concept fils to recognize tht ech enntiomer is seprte drug nd when products re licensed s the rcemic mixture they re products contining two drugs. Severl methods hve been reported for the enntioselective determintion of etodolc in phrmceuticl preprtions nd biologicl fluids. These methods included cpillry electrophoresis [12,13], cpillry electrochromtogrphy [14], gs chromtogrphy [15 17] nd high performnce liquid chromtogrphy [18 25] methods. An extensive literture survey reveled tht there re severl chromtogrphic methods for the determintion of etodolc in blood plsm, wheres, there re few other litertures disclosed only for the quntittive determintion of etodolc in phrmceuticl formultion smples. The reported methods were influenced by interference of endogenous substnces nd potentil loss of drugs in the re extrction procedure nd involving lengthy, tedious nd time consuming plsm smple preprtion nd extrction processes nd requiring sophisticted nd expensive instrumenttion. The development of new method cpble of determining drug mount in phrmceuticl dosge forms is importnt. Electronlyticl techniques hve been used for the determintion of wide rnge of drug compounds with the dvntges tht there re, in most, instnces no need for derivtiztion nd tht these techniques re less sensitive to mtrix effects thn other nlyticl techniques. Additionlly, ppliction of electrochemistry includes the determintion of electrode mechnism. Redox properties of drugs cn give insights into their metbolic fte or their in vivo redox processes or phrmcologicl ctivity [26 29]. Despite the nlyticl importnce of the electrochemicl behvior nd oxidtion mechnism of etodolc, no report hs been published on the voltmmetric study of the electrochemicl oxidtion of etodolc in nonqueous medi. It is well known tht the experimentl nd instrumentl prmeters directly ffect the electrochemicl process nd voltmmetric response of drugs. Consequently, it would be interest to investigte the oxidtion process of etodolc in protic medi. Therefore, the gol of this work ws the development of new liner sweep voltmmetry (LSV), squre wve voltmmetry (SWV) nd differentil pulse voltmmetry (DPV) methods for the direct determintion of etodolc in phrmceuticl preprtions without ny time consuming extrction or evportion steps prior to drug ssy. This pper describes fully vlidted simple, rpid, selective nd sensitive procedures for the determintion of etodolc employing LSV, SWV nd DPV methods the pltinum disc electrode. Besides, the methods were successfully pplied for the qulity control of three commercil etodolc tblets form to quntify the drug nd to check the formultion content uniformity. MATERIALS AND METHODS Etodolc ws obtined from Sigm (St. Louis, MO, USA). Acetonitrile (Fluk for HPLC nlysis) ws purified by drying with clcium hyride, followed by distilltion from phosphorus pentoxide. After purifiction in order to eliminte its wter content s much s possible, it ws kept over moleculr sieves. Lithium perchlorte (LiClO 4 ) ws purchsed from Fluk nd used s received without further purifiction. Etol, Tdolk nd Etodin tblets (200 mg etodolc) were obtined from phrmcy (Erzurum, Turkey). Electrochemicl instrumenttion: Electrochemicl experiments were performed on Gmry Potentiostt Interfce 1000 controlled with softwre PHE 200 nd PV 220. All mesurements were crried out in single comprtment 414 Indin Journl of Phrmceuticl Sciences July - August 2015

3 electrochemicl cell with stndrd three electrode rrngement. A pltinum disk with n re of 0.72 cm 2 nd pltinum wire were used s the working nd the counter electrodes, respectively. The working electrode ws successively polished with 1.0, 0.3 nd 0.05 µm lumin slurries (Buehler) on microcloth pds (Buehler). After ech polishing, the electrode ws wshed with wter nd sonicted for 10 min in cetonitrile. Then, it ws immersed into hot pirnh solution (3:1, H 2 SO 4, 30% H 2 O 2 ) for 10 min, nd rinsed copiously with wter. All potentils were reported versus Ag/AgCl/ KCl (3.0 M) reference electrode (BAS Model MF 2078) t room temperture. The electrolyte solutions were degssed with purified nitrogen for 10 min before ech experiment nd bubbled with nitrogen during the experiment. Operting conditions for SWV were pulse mplitude 25 mv, frequency 10 Hz, potentil step 4 mv; nd for DPV were pulse mplitude 50 mv, pulse width 50 ms, scn rte 100 mv/s. Preprtion of the stndrd nd qulity control solutions: The stock stndrd solution of etodolc ws prepred in 0.1 M LiClO 4 /cetonitrile to concentrtion of 100 µg/ml. Working stndrd solutions were prepred from the stock solution. Stndrd solutions were prepred s µg/ml for LSV, SWV nd DPV. The qulity control (QC) solutions were prepred by dding liquots of stndrd working solution of etodolc to finl concentrtions of 7.5, 25 nd 45 μg/ml for LSV, SWV nd DPV. Procedure for phrmceuticl preprtions: A totl 10 tblets of etodolc (Etol, Tdolk or Etodin) ws ccurtely weighed nd powdered. An mount of this powder corresponding to one tblet etodolc content ws weighed nd ccurtely trnsferred into 100 ml clibrted flsk nd 50 ml of 0.1 M LiClO 4 /cetonitrile ws dded nd then the flsk ws sonicted to 10 min t room tempture. The flsk ws filled to volume with 0.1 M LiClO 4 /cetonitrile. The resulting solutions in both the cses were filtered through Whtmn filter pper no 42 nd suitbly diluted to get finl concentrtion within the limits of linerity for the respective proposed methods. The drug content of etodolc tblets were clculted from the current potentil curves. RESULTS AND DISCUSSION The electrochemicl behvior of etodolc ws investigted t the Pt disc electrode in cetonitrile solution contining 0.1 M LiClO 4 s the supporting electrolyte by using cyclic voltmmetry (CV). Fig. 2 shows typicl cyclic voltmmogrm of 30 μg/ml etodolc recorded under these conditions for the scn rte of 0.1 V/s. In the nodic sweep, n oxidtion pek is seen t bout potentil of 1.03 V. Upon reversing the potentil scn, no reduction pek corresponding to this oxidtion wve is observed, indicting the irreversible nture of the electrode rections. In order to gin deeper insight into the voltmmetric wves, the effect of scn rte on the nodic pek currents (I m ) nd pek potentils (E p ) ws studied in the rnge of V/s of the potentil scn rtes in cetonitrile solution contining 30 μg/ml concentrtion of etodolc (fig. 3). The representtive liner sweep voltmmogrms obtined t Pt electrode for 30 μg/ml etodolc s function of the scn rte re presented in fig. 4. Scn rte dependency experiments show tht the pek currents for pek vry linerly with the scn rte (ν) (fig. 4 nd b), which points out the dsorption controlled process. However, the plots of logrithm of pek currents versus logrithm of scn rtes for 30 μg/ml concentrtion of etodolc disply stright lines with slope (fig. 4c), which re close to theoreticl vlue of 0.5 expected for n idel diffusion controlled electrode process [30]. log I m log ν curve is more eligible for this im, Fig. 2: Cyclic voltmmogrm. Cyclic voltmmogrm for the oxidtion of 30 μg/ml etodolc in cetonitrile contining 0.1 LiClO4 t Pt disk electrode, scn rte: 0.1 V/s. July - August 2015 Indin Journl of Phrmceuticl Sciences 415

4 therefore, diffusionl process for pek should be considered. These results suggest tht the redox species re diffusing freely from solution nd not precipitting onto the electrode surfce. The reson for this behvior my be due to the solubility of the Fig. 3: Liner sweep voltmmogrms. Liner sweep voltmmogrms for the oxidtion of 30 μg/ml etodolc in cetonitrile contining 0.1 M LiClO4 s function of scn rte. intermedite species in cetonitrile or poor dherence of products on the electrode surfce. As shown in fig. 3, the oxidtion pek potentil (E p ) for peks shift towrd more positive vlues with incresing scn rte. The reltionship between the pek potentil nd scn rte is described by the following eqution [31], 0' ( ) Ep = E + RT / 1 nf 1/2-1 ( s ) ( ) ln D k 0.5ln RT / 1 nf ( ) + RT / 1 nf / 2lnν nd from the vrition of pek potentil with scn rte αn cn be determined, where α is the trnsfer coefficient nd n is the number of electrons trnsferred in the rte determining step. According to this eqution, the plots of the pek potentils versus ln ν for oxidtion pek show liner reltionship (fig. 5). The slope indicte the vlue of αn is 0.61 for pek. Also, this vlue obtined indicte the totl irreversibility of the electron trnsfer b Fig. 4: Dependence of pek current on the scn rte (30 μg/ml). nd b re different scn rtes nd c is logrithmic scn rte. c 416 Indin Journl of Phrmceuticl Sciences July - August 2015

5 processes. This result show tht the chemicl step is fst following rection coupled to chrge trnsfer. The vlidtion ws crried out by estblishing specificity, linerity, ccurcy, precision, limit of detection (LOD), limit of quntifiction (LOQ), recovery, ruggedness ccording to ICH Q2B recommendtions [32,33]. The effects of common excipients nd dditives were tested for their possible interferences in the ssy of etodolc. The simulted nd plcebo smples were prepred nd nlyzed. It hs not been determined ny interference of these substnces t the levels found in dosge forms. Excipient tht ws used in this preprtion ws the most commonly used by the phrmceuticl industry. The presence of titnium dioxide, sodium chloride, tlc, lctose, strch, nd mgnesium sterte did not pper interfere in the results of the nlysis. Stndrd solutions were prepred s μg/ml (2.5, 5, 7.5, 10, 15, 20, 25, 30, 40 nd 50 μg/ml) for LSV, SWV nd DPV (figs. 6 8). Clibrtion curves were constructed for etodolc stndrd by plotting the concentrtion of compound versus pek current responses. The clibrtion curves were evluted by its correltion coefficients. The correltion coefficients (r) of ll the clibrtion curves were consistently greter thn The liner Fig. 5: Dependence of nodic pek potentils of voltmmetric pek for the oxidtion of 30 μg/ml etodolc on the scn rte. Fig. 6: Liner sweep voltmmogrms for different concentrtions of etodolc. Liner sweep voltmmogrms for different concentrtions of etodolc in cetonitrile solution contining 0.1 M LiCIO 4 (2.5, 5, 7.5, 10, 15, 20, 25, 30, 40 nd 50 μg/ml). Fig. 7: Squre wve voltmmogrms for different concentrtions of etodolc. Squre wve voltmmogrms for different concentrtions of etodolc in cetonitrile solution contining 0.1 M LiCIO 4 (2.5, 5, 7.5, 10, 15, 20, 25, 30, 40 nd 50 μg/ml). Fig. 8: Differentil pulse voltmmogrms for different concentrtions of etodolc. Differentil pulse voltmmogrms for different concentrtions of etodolc in cetonitrile solution contining 0.1 M LiCIO 4 (2.5, 5, 7.5, 10, 15, 20, 25, 30, 40 nd 50 µg/ml). July - August 2015 Indin Journl of Phrmceuticl Sciences 417

6 regression equtions were clculted by the lest squres method using Microsoft Excel progrm nd summrized in Tble 1. Accurcy of the ssy methods were determined for both intr dy nd inter dy vritions using the six times nlysis of the qulity control (QC) smples. Precision of the ssy ws determined by repetbility (intr dy) nd intermedite precision (interdy). Repetbility refers to the use of the nlyticl procedure within lbortory over short period of time tht ws evluted by ssying the QC smples during the sme dy. Intermedite precision ws ssessed by compring the ssys on different dys (2 dys). The intr dy ccurcy rnged from 0.37 to 1.85% nd precision from 1.58 to 4.69% (Tble 2). The results obtined from intermedite precision (inter dy) lso indicted good method precision. All the vlues were within the cceptnce criteri of 4.69%. The Limits of detection (LOD) nd quntifiction (LOQ) of etodolc by the proposed methods were determined using clibrtion stndrds. LOD nd LOQ vlues were clculted s 3.3 σ/s nd 10 σ/s, respectively, where S is the slope of the clibrtion curve nd σ is the stndrd devition of TABLE 1: LINEARITY OF ETODOLAC Method Rnge (µg/ml) LR R 2 LOD (µg/ml) LOQ (µg/ml) LSV y=100.51x SWV y=68.52x DPV y=24.635x Bsed on three clibrtion curves, LR: Liner regression, R 2 : coefficient of correltion, y: pek current, x: etodolc concentrtion, LOD: limit of detection, LOQ: limit of quntifiction, LSV: liner sweep voltmmetry, SWV: squre wve voltmmetry, DPV: differentil pulse voltmmetry y intercept of regression eqution (n=6) [33]. The LOD nd LOQ vlues of the methods were summrized in Tble 1. To determine the ccurcy of the LSV, SWV nd DPV methods nd to study the interference of formultion dditives, the recovery ws checked s three different concentrtion levels. Anlyticl recovery experiments were performed by dding known mount of pure drugs to pre nlyzed smples of commercil tblet forms. The recovery vlues were clculted by compring concentrtion obtined from the spiked smples with ctul dded concentrtions. These vlues re lso listed in Tble 3. In this study, the LSV, SWV nd DPV determintion of etodolc were crried out by different nlyst in sme instrument with the sme stndrd (Tble 4). The results showed no sttisticl differences between different opertors suggesting tht the developed method ws rugged. To evlute the stbility of etodolc, stndrd solutions were prepred seprtely t concentrtions covering the low, medium nd higher rnges of clibrtion curve for different temperture nd times. These solutions were stored t room temperture, refrigertory (4 ) nd frozen ( 20 ) temperture for 24 h nd 72 h. Stbility mesurements were crried out with LSV, SWV nd DPV methods. The results were evluted compring these mesurements with those of stndrds nd expressed s percentge devition nd etodolc ws found s stble t room temperture, 4 nd 20 for t lest 72 h. The stbility of etodolc ws obtined within the cceptnce rnge of %. Accurcy c Found±SD Precision TABLE 2: PRECISION AND ACCURACY OF ETODOLAC Method Added Intr dy Inter dy (µg/ml) Found±SD Precision Accurcy c RSD (%) b RSD (%) b LSV ± ± ± ± ± ± SWV ± ± ± ± ± ± DPV ± ± ± ± ± ± SD: Stndrd devition of six replicte determintions, b RSD: reltive stndrd devition, verge of six replicte determintions, c Accurcy: (percentge of reltive error) (found dded)/dded 100, LSV: liner sweep voltmmetry, SWV: squre wve voltmmetry, DPV: differentil pulse voltmmetry 418 Indin Journl of Phrmceuticl Sciences July - August 2015

7 TABLE 3: RECOVERY OF ETODOLAC IN PHARMACEUTICAL PREPARATIONS Phrmceuticl Added SWV DPV preprtion (µg/ml) Found±SD Recovery (%) RSD (%) Found±SD Recovery (%) RSD (%) Etol (5 μg/ml) 5 4.9± ± ± ± ± ± Tdolk (5 μg/ml) 5 4.8± ± ± ± ± ± Etodin (5 μg/ml) 5 5.1± ± ± ± ± ± RSD: Reltive stndrd devition, verge of six replicte determintions, SD: stndrd devition of six replicte determintions, SWV: squre wve voltmmetry, DPV: differentil pulse voltmmetry LSV, SWV nd DPV voltmmetry methods were pplied for the determintion of the commercil tblets (Tble 3). The results show tht high relibility nd reproducibility of three methods. The best results were sttisticlly compred using the F test. At 95% confidence level, the clculted F vlues do not exceed the theoreticl vlues (Tble 5). Therefore, there re no significnt difference between LSV, SWV nd DPV voltmmetry methods. The proposed methods were pplied to the direct determintion of etodolc in tblet dosge forms, using the clibrtion stright line without smple preprtion nd fter n dequte dilution. The drug ws recently included in United Sttes Phrmcopoei [34]. Etodolc tblets were lso determined with the officil procedure, which involves high performnce liquid chromtogrphic method [34]. Tble 5 compres the results of the nlysis of etodolc between the proposed nd officil methods. All methods showed similr ccurcy nd precision. According to the Student s t test, the clculted t vlues did not exceed the theoreticl vlue for significnce level of Sttisticl nlysis of the results showed no significnt difference between the performnce of the officil nd proposed methods s regrds to ccurcy nd precision. On the other hnd, the voltmmetric ssy is simple nd rpid compred with the high performnce liquid chromtogrphic ssy. Moreover, the ccurcy of the proposed methods ws lso evluted by recovery studies fter dding known mounts of the pure drug to vrious prenlysed formultions of etodolc nd pplying the procedure specified under experimentl section. As Tble 5 shows, good results demonstrte the TABLE 4: THE RESULTS OF ANALYSES OF ETODOLAC BY A DIFFERENT ANALYST Method Added (µg/ml) Found (µg/ml) men±sd Recovery (%) RSD (%) LSV 5 4.9± ± ± SWV 5 5.1± ± ± DPV 5 5.2± ± ± Men mesurements of six replicte determintions. RSD: Reltive stndrd devition, LSV: liner sweep voltmmetry, SWV: squre wve voltmmetry, DPV: differentil pulse voltmmetry TABLE 5: COMPARISON OF THE PROPOSED AND REPORTED METHODS FOR DETERMINATION OF ETODOLAC Prmeters LSV SWV DPV Officil method Men (recovery %) SD RSD (%) Vrince F test Theoreticl vlues t P=0.05, Ho hypothesis: No sttisticlly significnt difference exists between four methods, F t >F c : Ho hypothesis is ccepted (P>0.05). RSD: Reltive stndrd devition, SD: stndrd devition, LSV: liner sweep voltmmetry, SWV: squre wve voltmmetry, DPV: differentil pulse voltmmetry vlidity of the proposed methods for the determintion of etodolc in commercil dosge forms. The electrochemicl behvior of etodolc hs been studied in nonqueous medi by CV, LSV, SWV nd DPV voltmmetry methods. It hs concluded tht there is completely diffusion controlled current process which isn t ffected by dsorption phenomenon. Besides, in the present report, simple, rpid, sensitive, relible, specific, ccurte nd precise LSV, SWV nd DPV methods for the determintion July - August 2015 Indin Journl of Phrmceuticl Sciences 419

8 of etodolc in phrmceuticl preprtions were developed nd vlidted. The method described hs been effectively nd efficiently used to nlyze etodolc phrmceuticl tblets without ny interference from the phrmceuticl excipients. The voltmmetric run time of 3 min llows the nlysis of lrge number of smples in short period of time. Therefore, the methods cn be used effectively without seprtion for routine nlysis of etodolc in pure form nd its formultions. Finncil support nd sponsorship: Nil. Conflicts of interest: There re no conflicts of interest. REFERENCES 1. Bruke A, Smyth E, Fitgerld GA. Anlgesic ntipyretic gents; phrmcotherpy of gout. In: Brunton LL, Lzo JS, Prker KL, editors. Goodmn nd Gilmn s The Phrmcologicl Bsis of Therpeutics. New York: McGrw Hill; p Pirmohmed M, Jmes S, Mekin S, Green C, Scott AK, Wlley TJ, et l. Adverse drug rections s cuse of dmission to hospitl: Prospective nlysis of ptients. BMJ 2004;329: Mgill P, Ridgwy PF, Conlon KC, Nery P. 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