JESSY SHAJI *, DHANILA VARKEY

Transcription

1 Acdemic Sciences Interntionl Journl of Phrmcy nd Phrmceuticl Sciences ISSN Vol 4, Suppl 1, 2012 Reserch Article DEVELOPMENT OF A VALIDATED STABILITY-INDICATING HPTLC METHOD FOR DETERMINATION OF MELOXICAM IN BULK AND PHARMACEUTICAL FORMULATIONS: PERTINENCE TO ICH GUIDELINES JESSY SHAJI *, DHANILA VARKEY Deprtment of Phrmceutics, Prin. K.M. Kundni College of Phrmcy, Plot No. 23, Jote Joy Building, Rmbhu Slgonkr Mrg, Cuffe Prde, Mumbi , Indi. Emil: jshji@rediffmil.com ABSTRACT Received: 16 Sep 2011, Revised nd Accepted: 12 Nov 2011 A rpid, precise, selective nd sensitive, stbility-indicting HPTLC method for quntittive estimtion of Meloxicm (MLX) hs been developed nd vlidted. TLC luminium pltes (Merck) precoted with silic gel 60F254 ws used s the sttionry phse. The solvent system comprising of toluene: ethyl cette: methnol: glcil cetic cid in the rtio of 4:4:1.6:0.4 (v/v/v/v) gve dense compct spot with n Rf vlue of 0.61± 0.02, n=6. Densitometric nlysis ws crried out in reflectnce-bsorbnce mode t 358 nm. The method ws vlidted in complince with ICH Hrmonized Triprtite Guideline Q2 (R1) for linerity, limit of detection (LOD), limit of quntifiction (LOQ), precision, specificity, ccurcy, repetbility nd robustness. The drug response with respect to pek re ws liner over the concentrtion rnge ng/spot (n=6). The men (±SD) vlues of the slope, intercept nd correltion coefficient were (±0.089), (±24.516) nd (± ), respectively. The LOD nd LOQ were nd ng/spot respectively. Sttisticl evlution proved tht the estblished method ws ccurte, specific, precise, repetble nd robust for the estimtion of MLX. The degrdtion products were well resolved from the pure drug with significntly different Rf vlues nd thus cn be used to monitor stbility. Being simple nd economicl, the method cn be employed for the routine qulity control nlysis of MLX in bulk nd phrmceuticl dosge forms. Keywords: HPTLC; Meloxicm; Stbility-indicting; Degrdtion; Vlidtion; Bulk INTRODUCTION Meloxicm, [MLX; 4-hydroxy-2-methyl-N-(5-methyl-2-thizolyl) - 2H-benzothizine-3-crboxmide-1,1-dioxide)] is highly effective non-steroidl nti-inflmmtory drug (NSAID) of the enolic cid clss of oxicm derivtives 1. It hs the moleculr weight of Dlton 2 nd hs two pk vlues (pk1 = 1.09, pk2 = 4.18) 3. Its chemicl structure is shown in Fig.1. Fig. 1: Chemicl Structure of MLX MLX demonstrted high potency in niml tests for nti-rthritic ction, nd hs wider spectrum of nti-inflmmtory ctivity, combined with less gstric nd locl tissue irrittion thn NSAIDs vilble prior to its discovery. It is recurrently used to tret rheumtoid rthritis, osteorthritis, pucirticulr nd polyrticulr course juvenile rheumtoid rthritis. Besides its min therpeutic ppliction s n nti-inflmmtory nd nlgesic gent, it is lso rising s promising drug for the tretment of Alzheimer s disese nd cncer (minly colorectl nd denocrcinom) 4,5. Clinicl trils dt on MLX showed tht it hs superior gstrointestinl tolerbility, s would be predicted from its more selective inhibition of COX-2 reltive to COX-1 6. MLX is slowly but lmost completely bsorbed fter orl dministrtion with n bsolute biovilbility of 89%. Like mny other NSAIDs, MLX is prcticlly insoluble in wter. MLX cn be grded in Clss II, of the Biophrmceuticl Clssifiction System (BCS), which mens low queous solubility nd rpid bsorption (high permebility) through the gstrointestinl trct 7. MLX is strongly bound to plsm proteins (99.5%) nd displys liner phrmcokinetics. Pek plsm concentrtions re reched 5-6 h fter orl dosing when tken concomitntly with light mel, s generlly recommended. It hs fvorble tolerbility profile nd higher therpeutic index thn tht of other NSAIDs, including piroxicm, diclofenc nd indomethcin 8. MLX undergoes extensive metbolism, primrily by cytochrome P450 CYP2C9 nd to minor extent by CYP3A4, forming four mjor inctive metbolites. The phrmcokinetics of MLX is liner over the dose rnge mg nd remins unchnged from single to multiple dosing. Totl MLX clernce is 7-8 ml min -1 nd the terminl elimintion hlf-life is pproximtely 20 h, mking MLX suitble for once dily dosing. Stedy-stte drug plsm concentrtions re reched within 3-5 dys 9. Although MLX is one of the most widely used nti-inflmmtory drugs, no reference procedure exists for its determintion in phrmceuticl formultions in Interntionl Phrmcopoeis. Effectively, there is only one monogrph for MLX in the British Phrmcopoei, bsed on non queous titrtion, which is not pplicble to tblets due to interference from excipients used in phrmceuticl formultions 10. Few methods hve been reported for the determintion of MLX including non queous titrtion 11, spectrophotometric methods 12,13, high performnce liquid chromtogrphy (HPLC) methods 14-17, fluorimetric methods 18,19, turbidimetric method 20, densitometric method 21, electrochemicl method 22, voltmmetric method 23, chemiluminometric 24 nd electrophoretic method 25. However most of these nlyticl methods hve some limittions for routine nlysis such s tedious nd time consuming smple preprtion, constnt dependency on opertor, long smple nlysis time nd use of expensive solvents nd pprtus. In view of this, high performnce thin lyer chromtogrphy (HPTLC) bsed methods could be considered s good lterntive, s they re being explored s n importnt tool in routine drug nlysis. A mjor dvntge of HPTLC is its bility to nlyze severl smples simultneously using smll quntity of mobile phse, thereby reducing the time nd cost of nlysis 26. HPTLC cn be used for the nlysis of lrge s well s smll smples 21. Furthermore for such methods extrction procedure is not lwys required nd could be used for nlyzing drug without ny interference from excipients. The prent drug stbility guidelines [Q1A (R), 2000] 27 issued by ICH suggest tht stress studies should be conducted on drug to estblish its inherent stbility chrcteristics. Stress degrdtion studies re necessry to develop nd demonstrte specificity of stbility-indicting methods nd to determine the degrdtion pthwys nd degrdtion products of the ctive ingredients. They

2 cn be functionl in the investigtion of the chemicl nd physicl stbility of crystl forms, stereochemicl stbility nd differentiting drug substnce-relted degrdtion products in formultions, thereby supporting the suitbility of the proposed nlyticl procedures. It lso requires tht nlyticl test procedures for stbility smples should be fully vlidted nd the ssys should be stbilityindicting 28. Accordingly, the objective of this work ws to subject MLX to vriety of stress test conditions to estblish intrinsic stbility of the drug nd to develop vlidted stbility method. The present study reports development of n ccurte, specific, repetble nd stbility indicting method for determintion of MLX in presence of its degrdtion products nd relted impurities for ssessment of purity of bulk drug nd stbility of its phrmceuticl dosge form. MATERIALS AND METHODS Mterils Phrmceuticl grde of MLX ws obtined s gift smple from Zest Phrm, Indore, Indi (Btch No. ALC/MLX/090302). It ws certified to contin 99.78% w/w (on dried bsis) nd ws used without further purifiction. All chemicls nd regents used were of nlyticl grde nd were purchsed from Merck Chemicls, Mumbi, Indi. Commercil phrmceuticl preprtion Muver 15 (Sun Phrm, Sikkim) contining MLX 15mg ws purchsed from the locl phrmcy. All dilutions were performed in clibrted volumetric flsks. Instrumenttion Linomt V smple pplictor (Cmg, Switzerlnd) with 100 µl smple syringe (Hmilton, Bonduz, Switzerlnd), twin trough glss chmber (20cm x 10cm, Cmg, Switzerlnd), TLC plte heter (Cmg, o C), Cmg TLC scnner (4) with wincats softwre (version 1.4.5), TLC visulizer (Cmg), UV inspection cbinet (Cmg), Mettler nlyticl blnce AB 104-S/FACT (Mettler Toledo) (ccurcy ±0.01mg) nd ultrsonic bth sonictor (PCI Anlytics, Mumbi). Chromtogrphic HPTLC conditions Experimentl nlysis ws performed on silic gel 60F254 HPTLC pltes (20cm x 10cm with 250 µm thickness; E Merck, Drmstdt, Germny, Btch-HX011551) using mobile phse consisting of toluene: ethyl cette: methnol: glcil cetic cid in the rtio of 4:4:1.6:0.4 (v/v/v/v). Prior to chromtogrphic nlysis the pltes were wshed in methnol, dried in current of dry ir nd ctivted t 110 o C for 5 min. Smples were spotted in the form of bnds of width 8mm with Cmg microlitre syringe. A constnt ppliction rte of 150nl/s ws used nd the spce between two bnds were 5mm. Monochromtor bnd width ws set t 20nm, ech trck ws scnned six times nd bseline correction ws used. Liner scending development ws crried out in 20cm x 10cm twin trough glss chmber sturted with mobile phse. The optimized chmber sturtion time for the mobile phse ws 15 min t room temperture (26±2 o C) nd reltive humidity (50±5%). No neckless effect ws seen during the development nd it took pproximtely 20 min for the complete development of the TLC plte. The length of ech chromtogrm run ws 80mm. Consequent to development, the pltes were dried in current of ir by use of n ir dryer. Densitometric scnning ws performed in the reflectncebsorbnce mode t 358nm, operted by Cmg wincats softwre (V 1.4.5). The source of rdition used ws deuterium lmp emitting continuous UV spectrum between 190 nd 400nm. The slit dimension ws kept t 6mm x 0.30mm nd the scnning speed ws 20mm/s. Concentrtions of the compound chromtogrphed were determined from the intensity of the diffused light. Evlution ws by pek res with liner regression. Scnned pek res were recorded for ech smple t ech concentrtion level. The verge pek res nd vritions in pek re obtined were expressed s percent reltive stndrd devition (% RSD). Clibrtion plot of MLX A stock solution of MLX (1mg/ml) ws prepred in chloroform. 0.5 ml of stock solution ws further diluted to 10 ml with chloroform to get stndrd solution of 0.05mg/ml. Approprite volumes of stndrd solution were spotted to obtin MLX in the concentrtion rnge of ng (n=6). The plte ws developed nd scnned s described bove. Well resolved compct bnd of drug ws scnned t 358nm. The linerity plot ws obtined by plotting verge pek re t ech concentrtion ginst corresponding bnd concentrtions of MLX (ng/spot). Liner regression nlysis ws employed to clculte the regression equtions nd the correltion coefficients. Method Vlidtion The proposed method ws vlidted in complince with ICH Guidelines. The method ws vlidted for linerity nd rnge, limit of detection (LOD), limit of quntittion (LOQ), precision, specificity, ccurcy, repetbility nd robustness. Limit of detection nd limit of quntittion Bsed on the stndrd devition of the response nd the slope, LOD nd LOQ were estimted using the formule: LOD= 3.3 σ/s Where σ = the stndrd devition of the response S = the slope of the clibrtion curve LOQ = 10 σ/s Where σ = the stndrd devition of the response S = the slope of the clibrtion curve LOD nd LOQ were determined from the stndrd devitions of the responses for six replicte determintions. Precision Precision is mesure of the reproducibility of the nlyticl method under norml operting conditions. Precision is expressed s reltive percent stndrd devition (% RSD). The intr- nd inter-dy precisions were crried out t three different concentrtion levels, 300, 500 nd 700 ng/spot (n=6) respectively. Repetbility of mesurement of pek re nd % content cn be defined s the precision of the method when repeted under sme operting conditions (sme equipments, regents, settings nd lbortory) over short intervl of time. Repetbility ws ssessed by chromtogrphy of six replictes of 100% of the test concentrtion (500ng/spot) nd expressed in terms of coefficient of vrition (CV). Selectivity nd specificity Selectivity of n nlyticl method is its cpbility to determine precisely nd specificlly the nlyte in the presence of components tht my be expected to be present in the smple mtrix like mtrix components, impurities or degrdtion products. UV spectrum of stndrd drug nd smple t selected wvelength ws compred. Specificity ensures tht the signl mesured comes from the nlyte of interest nd tht there is no interference from excipients, impurities or degrdtion products. Specificity ws determined by nlyzing stndrd drug nd test smple. The spot for MLX in the smple ws confirmed by compring the Rf nd spectrum of the spot with tht of the stndrd. The pek purity of MLX ws judged by compring the spectrum t three different regions of the spot i.e. pek strt, pek pex, nd pek end positions of the spot. Accurcy (Recovery studies) Accurcy of n nlyticl method is the closeness of test results to true vlue. It ws determined by the ppliction of nlyticl procedure to recovery studies, where known mount of stndrd is spiked in prenlyzed smple solutions. The mount of drug recovered in ccurcy study ws in the rnge, which indicted tht the method is ccurte. Accurcy ws studied t different levels i.e. 0%, 80%, 100% nd 120% by ddition of pure drug to previously nlyzed test smples. The mixtures were renlyzed by the proposed method. The experiment ws conducted in triplicte nd percent recovery ws clculted. 161

3 Robustness The robustness of n nlyticl method my be defined s the mesure of its bility to remin unchnged by smll, but purposeful vritions in the method prmeters nd provides n indiction of its dependbility during norml usge. The robustness of the method ws determined by vritions in mobile phse composition (±0.2ml), volume of mobile phse (±2ml), chmber sturtion period (±5min), development distnce (±0.5cm), nd time from development to scnning (0, 10, 20 nd 30 min). One fctor t time ws chnged t concentrtion level of 400ng/bnd (n=6) of MLX, to study the effect on pek res of the drug. Anlysis of mrketed formultion Mrketed tblet formultions (20 tblets, lbel clim 15mg/tblet) were weighed nd powdered. Tblet triturte equivlent to the lbel clim of the drug ws dissolved in chloroform nd subjected to soniction for 10 min. The volume ws mde up to 100 ml nd filtered through Whtmn filter pper. The filtrte obtined ws suitbly diluted nd spotted onto the plte followed by development nd scnning s described in section chromtogrphic HPTLC conditions. The nlysis ws repeted in triplicte. Any possibility of excipients interference ws lso studied. Forced degrdtion studies A stock solution contining 10 mg/ml MLX ws prepred. This solution ws used for stress degrdtion to indicte specificity of the proposed nlyticl method nd its stbility indicting property. In ll the degrdtion studies the verge pek re of three replictes ws obtined. Acid nd bse induced degrdtion studies Decomposition studies were crried out by refluxing 5ml of drug solution with 5ml ech of 1N HCl nd 1N NOH. The mixtures were refluxed for 6h respectively t 60 o C. The forced degrdtion in cidic nd lkline medi ws crried out in drk to exclude the possible degrdtion effect of light. The solutions (0.5ml) were tken nd neutrlized nd subsequently diluted to 10 ml with chloroform. The resulting solutions were pplied to TLC plte in such wy tht the finl concentrtion ttined ws 5000ng/spot for both cid nd bse degrdtion products nd the chromtogrms were run s described in previous section. Dry het nd wet het degrdtion The stndrd drug ws stored in oven t 50 o C for 72h to study dry het degrdtion nd for wet degrdtion drug ws stored t 50 o C nd 75% reltive humidity for 3 months. Photo degrdtion The photochemicl stbility of the drug ws studied by exposing the stock solution to direct sunlight for 7 dys. After suitble dilution, concentrtion of 5000ng/spot ws pplied nd the chromtogrms were run s described in previous section. Oxidtive degrdtion (Hydrogen peroxide induced) Studies were performed in 3% (v/v) nd 30% (v/v) hydrogen peroxide t room temperture for 48h respectively. The mixture ws stored in drk to exclude the possible effect of light. The solution ws then heted in boiling wter bth for 10 min to completely remove excess of hydrogen peroxide. The resultnt solutions were pplied to TLC plte to chieve finl concentrtion of 5000ng/spot nd the chromtogrms were run s described in previous section. Detection of the relted impurities Relted impurities were determined by spotting higher concentrtions of the drug with n im to detect nd quntify them. MLX (50 mg) ws dissolved in 5ml of chloroform to ttin concentrtion of 10mg/ml nd this solution ws termed s smple solution. 0.5 ml of the smple solution ws diluted to 100ml with chloroform to get stndrd solution (0.05mg/ml). 1 µl of smple solution (10,000ng/spot) nd 10µl of stndrd (500ng/spot) were pplied on the TLC plte nd the chromtogrms were run s described in previous section. RESULTS AND DISCUSSION Development of optimum mobile phse The HPTLC method ws optimized with n im to develop stbility-indicting ssy method. During mobile phse optimiztion vrious mobile phse compositions including toluene: methnol (8:2), toluene: methnol: glcil cetic cid (8:1.5:0.5), toluene: ethyl cette: methnol (4:4:2) nd different proportions of toluene: ethyl cette: methnol: glcil cetic cid were tried. Both pure drug s well s forced degrdtion smples were spotted onto precoted TLC pltes nd tried with the fore mentioned mobile phse systems. Higher concentrtions of toluene resulted in low Rf vlues of Incresing the solvent strength (ε o ) of the mobile phse decreses retention nd increses the Rf vlue. As result ethyl cette ws used in equl proportions s toluene long with methnol to hve considerble ε o. The spot obtined ws diffused with n Rf vlue of Glcil cetic cid ws used in the mobile phse s modifier to reduce the diffusion of the spots. Amongst the combintions tried toluene: ethyl cette: methnol: glcil cetic cid (4:4:1.6:0.4, v/v/v/v) ws finlized s the mobile phse. The spots developed were dense nd compct with n Rf vlue of 0.61± Typicl pek of MLX is shown in Fig.2. The pek obtined ws shrp nd symmetricl in nture. The spectrum obtined fter densitometric scnning showed pek t 358 nm nd is shown in Fig 3. Vlidtion prdigms Linerity The linerity of n nlyticl method is its bility, within given rnge, to obtin test results which re either directly or through mthemticl trnsformtion, proportionl to concentrtion of the nlyte in the smple. The method ws found to be liner (r 2 = ± ) in the concentrtion rnge of ng/spot (n=6), with respect to pek re. Pek re nd concentrtion ws subjected to liner regression nlysis to determine clibrtion eqution nd correltion coefficients. No significnt difference ws observed in the slopes of stndrd curves (ANOVA, P< (P vlue, one smple t test). The three dimensionl overly of densitogrms of the clibrtion spots of MLX t 358nm is displyed in Fig.4. The regression dt is reported in Tble 1. Tble 1: Liner regression dt for the clibrtion curves Prmeters Vlue Detection Wvelength (nm) 358 Beer s Lw Limit (ng/bnd) Correltion Coefficient (r 2 ± SD) ± Intercept (c) ± SD ± Confidence limit of intercept b Slope (m) ± SD ± 0.08 Confidence limit of slope b SD of residuls from line (Sy.x) n=6; b Confidence intervl: 95% 162

4 [ [ Shji et l. Fig. 2: Densitogrm of MLX (Rf: 0.61±0.02), Wvelength: 358nm, Mobile phse: toluene: ethyl cette: methnol: glcil cetic cid (4:4:1.6:0.4, v/v/v/v) Fig. 3: Spectrum of MLX mesured from nm Fig. 4: Three dimensionl overly of densitogrms of the clibrtion spots of MLX t 358nm Tble 2: Sttisticl evlution of precision (repetbility) of developed method Drug: MLX Repetbility Conc. (ng/spot) Are ± SD % Content ± SD Coefficient of Vrition Pek Are % Content ± ± n=5 Tble 3: Summry of Intr-dy nd Inter-dy method precision Amount (ng/spot) Intr-dy precision by pek re Inter-dy precision by pek re Men ± SD % Content ± SD %RSD Men ± SD % Content ± SD %RSD ± ± ± ± ± ± ± ± ± ± ± ± n=5 163

5 LOD nd LOQ The LOD nd LOQ were found to be nd ng respectively. Precision The repetbility of mesurement of pek re nd % content ws expressed s CV nd found to be nd for MLX (Tble 2). Results for intr- nd inter-dy vrition studied t three levels 300, 500 nd 700 ng/spot is shown in Tble 3. The % RSD ws found to be <2% in ll cses nd thus indicte tht the method is highly precise. Specificity nd selectivity The method ws selective nd specific for the determintion of MLX. Good correltion (r=0.9999) ws obtined between the stndrd nd smple spectr of MLX. The overly spectrum of stndrd nd smple is shown in Fig. 5. A single spot with n Rf vlue of 0.61 ws observed in MLX stndrd nd smples from mrketed MLX tblet. In degrdtion studies the spots of degrded products were well resolved from the drug spot. Fig. 5: Spectrl overly of stndrd nd smple (formultion) Accurcy (Recovery studies) The proposed method for extrction nd estimtion of MLX from phrmceuticl dosge form fter spiking with 80%, 100% nd 120% showed good recoveries in the rnge of % s listed in Tble 4. Robustness Robustness ws studied by determining the effect of smll vritions in the mobile phse composition, mobile phse volume, chmber sturtion time, migrtion distnce nd time from chromtogrphy to scnning. The % RSD of pek res ws clculted for ech vrible nd ws found to be less thn 2%. The low vlues of % RSD s listed in Tble 5 indicte tht the method is robust. Solution stbility Solution of 100% test concentrtion (500ng/spot) ws stored t room temperture for 6, 12, 24, 48 nd 72h, respectively. The chromtogrphic nlysis ws performed on the sme TLC plte. After development, the chromtogrm ws studied for dditionl spots if ny. The pek re nd % RSD ws found to be ± nd 1.81 respectively. No indiction of instbility of the nlyte ws found in the smple solution. Anlysis of mrketed formultion A single nd compct spot t Rf vlue of 0.61 ws observed in the chromtogrm of the smples extrcted from tblets. No interference from the excipients ws found. The drug content ws found to be % ± 1.15, with 1.14 % RSD. MLX ws stble in the mrketed formultion nlyzed by this method nd no degrdtion hd occurred. Acceptble limit of % RSD vlue indicted the suitbility of the proposed method for routine nlysis of MLX in phrmceuticl dosge form. Forced Degrdtion (Stbility indicting property) Acid nd bse induced degrdtion product The rte of degrdtion in cid ws slower s compred with lkli. The chromtogrm of the cid degrded smple showed pek t Rf vlue of 0.16 (Fig.6). For bse degrded smple, pek t Rf vlue of 0.16 nd 0.69 were observed (Fig.7). The results indicte tht MLX undergoes degrdtion under cidic nd bsic conditions. Tble 4: Recovery studies of MLX Recovery Level Initil mount (ng) MLX dded (ng) Totl mount (ng) MLX found (ng) ± SD % Recovery ± SD ± ± ± ± ± ± ± ± 3.29 n=3 Tble 5: Results of Robustness studies Prmeter Men Rf %RSD of pek re Mobile phse composition (±0.2ml) 0.59 ± Mobile phse volume (±2ml) 0.60 ± Chmber sturtion time (±5 min) 0.61 ± Migrtion distnce (±0.5 cm) 0.60 ± Time from chromtogrphy to scnning (±10 min) 0.59 ± n=6 164

6 Fig. 6: Chromtogrm of cid induced degrdtion; pek 1(degrdtion product) (Rf: 0.16), pek 2 (MLX) (Rf: 0.61) Fig. 7: Chromtogrm of bse induced degrdtion; pek 1, 3 (degrdtion product) (Rf: 0.18, 0.69), pek 2 (MLX) (Rf: 0.62) Hydrogen peroxide induced degrdtion product The smple showed n dditionl pek with 3% (w/v) (Fig.8) nd 30% (w/v) t Rf vlue of 0.16 (Fig.9). The drug ws considerbly stble t room temperture. Fig. 8: Chromtogrm of hydrogen peroxide (3%) induced degrdtion; pek 1(degrdtion product) (Rf: 0.16), pek 2(MLX) (Rf: 0.61) 165

7 Fig. 9: Chromtogrm of hydrogen peroxide (30%) induced degrdtion; pek 1(degrdtion product) (Rf: 0.16), pek 2(MLX) (Rf: 0.61) Dry nd wet degrdtion product No dditionl peks were observed (Fig.10, 11). MLX ws stble under these conditions showing negligible degrdtion to dry nd wet het degrdtion. Fig. 10: Chromtogrm of dry het induced degrdtion; pek 1 (MLX) (Rf: 0.61). Fig. 11: Chromtogrm of wet het degrdtion; pek 1 (MLX) (Rf: 0.61) 166

8 Photolytic degrdtion product An dditionl pek ws observed t Rf vlue of 0.17 (Fig.12), however % recovery of MLX showed negligible degrdtion. The results for forced degrdtion studies re listed in Tble 6. Fig. 12: Chromtogrm of photo degrded MLX pek 1(degrdtion product) (Rf: 0.17); pek 2 (MLX) (Rf: 0.61) Tble 6: Forced Degrdtion of MLX Degrdtion Condition % Recovery Rf vlue of degrdtion products Acid 1N HCl Bse 1N NOH , 0.69 Oxidtive 3% H2O Oxidtive 30% H2O Dry het (50 o C,72h) Wet het (50 o C, 75% RH) Photolytic (sunlight, 7dys) Refluxed Detection of relted impurities The chromtogrphic nlysis reveled tht the smple showed one dditionl spot t Rf vlue of 0.16 (Fig.13). The re of the dditionl spot (1702.8) ws found to be much less s compred to the min spot ( ) from the stndrd solution. From the degrdtion results it cn be observed tht the Rf vlue of cid, bse, oxidtive nd photo degrdtion components equl with the impurity present in the drug. However bse induced degrdtion showed n dditionl degrdtion component eluted t Rf vlue of 0.69, which ws not detected in the present determintion. The spectr recorded for both smple nd impurity is shown in Fig.14. The dt of summry of vlidtion prmeters re listed in Tble 7. Fig. 13: Chromtogrm of MLX nd its impurity; pek 1 impurity, Rf: 0.16: pek 2 MLX, Rf:

9 Fig. 14: Spectr Comprison: MLX nd impurity Tble 7: Summry of vlidtion prmeters Prmeter Dt Linerity rnge Correltion coefficient ± Regression eqution Y= 8.192x Limit of detection ng/spot Limit of quntifiction ng/spot Accurcy ± 0.25 Precision Repetbility of ppliction (n=5) CV: nd for pek re nd % content respectively Inter-dy (n=3) Intr-dy (n=3) Robustness Robust Specificity Specific CONCLUSION Sttisticl nlysis of the dt estblishes tht the developed HPTLC method is specific, ccurte, precise nd stbility-indicting. The vlidted method is suitble for nlysis of MLX in both bulk nd phrmceuticl dosge form without ny interference from the excipients. The method cn be employed to determine the purity of the drug by detecting ny relted impurities present. The method is efficient in seprting the degrdtion components from the min nlyte MLX, nd hence cn be considered s stbility-indicting. This nlyticl method my be extended for determintion of MLX in plsm nd other biologicl fluids nd wrrnt further studies. ACKNOWLEDGEMENT The uthors re thnkful to Zest Phrm, Indore, Indi for gift smple of MLX, UGC for Finncil ssistnce nd Reserch Fellow nd Anchrom Lbortories, Mumbi for technicl support. REFERENCES 1. Ah YC, Choi JK, Choi YK, Ki HM, Be JH. A novel trnsderml ptch incorporting meloxicm: in vitro nd in vivo chrcteriztion. Int. J. Phrm. 2010; 385: Mhmood KT, Khn B, Ashrf M, Hq IU. Specific nd simple HPLC ssy of ecofriendly meloxicm in phrmceuticl formultions. Journl of Phrmceuticl Sciences nd Reserch 2010; 2(12): Luger P, Dneck K, Engel W, Trummlitz G, Wgner K. Structure nd physicochemicl properties of meloxicm, new NSAID. Eur. J. Phrm. Sci. 1996; 4: Ambrus R, Kocbek P, Kristl J, Sibnc R, Rjko R, Revesz PS. Investigtion of preprtion prmeters to improve the dissolution of poorly wter-soluble meloxicm. Int. J. Phrm. 2009; 381: Bndrkr FS, Vvi PR. A stbility indicting HPLC method for the determintion of meloxicm in bulk nd commercil formultions. Tropicl Journl of Phrmceuticl Reserch 2009; 8(3): Mrtin RM, Bisws P, Mnn RD. The incidence of dverse events nd risk fctors for upper gstrointestinl disorders ssocited with meloxicm use mongst ptients in generl prctice in Englnd: cohort study. Br. J. Clin. Phrmcol. 2000; 50: Vignduzzo SE, Cstellno PM, Kufmn TS. Development nd vlidtion of dissolution test for meloxicm nd pridinol mesylte from combined tblet formultion. Indin J. Phrm. Sci. 2010, 72(2): Mhmood KT, Ashrf M. Absolute biovilbility of orl meloxicm in helthy dogs. The Journl of Animl & Plnt Sciences 2010; 20(3): Meineke I, Turck D. Popultion phrmcokinetic nlysis of meloxicm in rheumtoid rthritis ptients. Br. J. Clin. Phrmcol. 2002; 55: Vsiliki V, Pinto PCAG, Luci M, Sriv MFS, Lim JLFC. Sequentil injection determintion of meloxicm in phrmceuticl formultions with spectrophotometric detection. Cn. J. Anl. Sci. Spectros. 2007; 52(6): Zwill NH, Mohmmd MAA, Kousy NME, Aly SMEM. Determintion of meloxicm in bulk nd phrmceuticl formultions. J. Phrm. Biomed. Anl. 2003; 32:

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