SENSITIVE SPECTROPHOTOMETRIC DETERMINATION OF ACECLOFENAC FOLLOWING AZO DYE FORMATION WITH 4-CARBOXYL-2,6-DINITROBENZENE DIAZONIUM ION

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1 Act Polonie Phrmceutic ñ Drug Reserch, Vol. 69 No. 2 pp. 203ñ211, 2012 ISSN Polish Phrmceuticl Society SENSITIVE SPECTROPHOTOMETRIC DETERMINATION OF ACECLOFENAC FOLLOWING AZO DYE FORMATION WITH 4-CARBOXYL-2,6-DINITROBENZENE DIAZONIUM ION SEGUN A. ADERIBIGBE, OLAJIRE A. ADEGOKE*, OLAKUNLE S. IDOWU nd SEFIU O. OLALEYE Deprtment of Phrmceuticl Chemistry, Fculty of Phrmcy, University of Ibdn, Orit UI, Ibdn, Nigeri Abstrct: The study is description of sensitive spectrophotometric determintion of ceclofenc following zo dye formtion with 4-crboxyl-2,6-dinitrobenzenedizonium ion (CDNBD). Spot test nd thin lyer chromtogrphy reveled the formtion of new compound distinct from CDNBD nd ceclofenc. Optimiztion studies estblished rection time of 5 min t 30 o C fter vortex mixing the drug/cdnbd for 10 s. An bsorption mximum of 430 nm ws selected s nlyticl wvelength. A liner response ws observed over 1.2ñ4.8 µg/ml of ceclofenc with correltion coefficient of nd the drug combined with CDNBD t stoichiometric rtio of 2 : 1. The method hs limit of detection of µg/ml, limit of quntittion of 1.22 µg/ml nd is reproducible over three dy ssessment. The method gve Sndellís sensitivity of ng/cm 2. Intr- nd inter-dy ccurcies (in terms of errors) were less thn 6% while precisions were of the order of 0.03ñ1.89 % (RSD). The developed spectrophotometric method is of equivlent ccurcy (p > 0.05) with British Phrmcopoei, 2010 potentiometric method. It hs the dvntges of speed, simplicity, sensitivity nd more ffordble instrumenttion nd could found ppliction s rpid nd sensitive nlyticl method of ceclofenc. It is the first described method by zo dye derivtiztion for the nlysis of ceclofenc in bulk smples nd dosge forms. Keywords: ceclofenc tblets, CDNBD, zo dye derivtiztion, spectrophotometric nlysis Chemiclly, ceclofenc is 2-(2-{2-[(2,6- dichlorophenyl)mino]phenyl}cetyl)oxycetic cid. It is n nlogue of diclofenc belonging to the clss of non-steroidl nti-inflmmtory drugs. It is commonly vilble in tblet form. The British Phrmcopoei stted its purity to be between 99.0 to 101% (1). Aceclofenc is phenylcetic cid derivtive. It occurs s white or lmost white, crystlline powder. It hs melting point of 149ñ150 O C (2). It is prcticlly insoluble in wter, freely soluble in cetone, soluble in ethnol (96 percent). Aceclofenc is potent inhibitor of cyclooxygense enzyme which is involved in the production of prostglndins. It lso inhibits the synthesis of meditors of inflmmtory response e.g., cytokines, interleukin, tumor necrosis fctor nd PGE 2 (3). Action of ceclofenc hs been found to be due to stimultion of synthesis of interleukin receptor ntgonist nd decrese of production of nitric oxide in humn rticulr chondrocytes subjected to inflmmtory stimuli (4). Aceclofenc is well bsorbed from the gstrointestinl trct. Pek plsm concentrtion is reched in 1ñ3 h fter orl dose. It is more thn 99% bound to plsm proteins. The plsm t 1/2 is bout 4 h. About two-third of dose is excreted in the urine s hydroxymetbolites (5). The drug is used for the relief of pin nd inflmmtion ssocited with rheumtoid rthritis, osteorthritis nd nkylosing spondylitis. Vrious methods hve been reported in the literture for the ssy of ceclofenc either lone or in combined form in drug formultions. The nlyticl techniques include: spectrophotometric colorimetric, spectrofluorimetric, densitometric, HPLC, RP- HPLC nd titrimetric. El-Moghzy et l. (6) reported the ssy of ceclofenc in pure form nd in phrmceuticl formultion by spectrophotometric method using p-dimethylminocinnmldehyde in cidified methnol to give stble colored complex fter heting t 75 O C for 20 min. Absorption mesurements were crried out t nm (6). Also, El- * Corresponding uthor: e-mil: jirede@yhoo.com; phone:

2 204 SEGUN A. ADERIBIGBE et l. Kousy demonstrted tht ceclofenc in combintion with etodolc could be ssyed by the formtion of colored complexes between the drugs nd p- dimethylminobenzldehyde regent in the presence of sulfuric cid nd ferric chloride. Mesurement of the bsorbnces ws crried out t nd nm for etodolc nd ceclofenc, respectively (7). Colorimetric estimtion of ceclofenc nd indpmide ws shown by Shingvi nd Goyl (8) using Folin-Cioclteu (FC) s chromogenic regent. The method is bsed on the formtion of blue nd green colored products. Aceclofenc shows bsorbnce mxim t nm nd linerity in the concentrtion rnge of 80ñ160 µg/ml (8). Shh et l. (9) reported vlidted spectrophotometric ssy of ceclofenc in tblet formultion by mesuring the UV bsorbnce in phosphte buffer of ph 7.4. The bsorption mximum of ceclofenc in the buffer ws 273 nm t the different dilutions prepred (20 nd 1000 µg/ml). Vrious stbility-indicting spectrophotometric methods for the determintion of ceclofenc in the presence of its min degrdtion product, diclofenc hve lso been described. Three methods were described by Hsn et l. Method A utilizes third derivtive spectrophotometry t 242 nm. Method B is the spectrophotometric method bsed on the simultneous use of the first derivtive of rtio spectr nd mesurement t 245 nm. Method C is ph-induced difference spectrophotometry using UV mesurement t 273 nm. Regression nlysis of Beerís plot showed good correltion in the concentrtion rnges 50, 100 nd 150 µg/ml for methods A, B, nd C, respectively (10). In similr work (11) two methods were developed for the determintion of ceclofenc. In the first method, third-derivtive spectrophotometry (D 3 ) ws used. The D 3 bsorbnce ws mesured t 283 nm where the hydrolytic degrdtion product ñ diclofenc, does not interfere. The suggested method shows liner reltionship in the rnge of 4ñ24 µg/ml with men percentge ccurcy of ± This method determines the intct drug in the presence of up to 70% of degrdtion product with men percentge recovery of ± The second method depends on Rtio-Spectr first-derivtive (RSD 1 ) spectrophotometry t 252 nm for ceclofenc nd t 248 nm for determintion of degrdtion product over concentrtion rnges of 4ñ32 µg/ml for both ceclofenc nd diclofenc with men percentge ccurcy of ± 0.84 nd ± 0.72 for pure drugs nd ± nd ± 0.74 for lbortory-prepred mixtures, respectively (11). A number of chromtogrphic nd densitometric techniques hd been employed in the determintion of ceclofenc in the biologicl system, bulk nd phrmceuticl formultions. A stbility-indicting ssy of ceclofenc by HPLC nd densitometry in the presence of its degrdtion products over concentrtion rnge of 20ñ70 µg/ml nd 1ñ10 µg per spot nd men recoveries of ± 0.90 nd ± 1.09 ws described (6). Simultneous determintion of ceclofenc, prcetmol nd chlorzoxzone by RP-HPLC ws described by Mngonkr et l. (12). In simultneous determintion of ceclofenc, prcetmol nd tiznidine (13) chromtogrphic seprtion of the three drugs were performed on Hypersil C 18 column (250 mm 4.6 mm, 5 µm) s sttionry phse. RP-HPLC method developed by Hsn et l. depended on using ethnol : wter (60:40, v/v) s mobile phse t flow rte of 1 ml/min nd UV detection t 275 nm. Regression nlysis of Beerís plot reveled good correltion in the concentrtion rnge 1ñ50 µg/ml (10). Other methods include densitometric method for nlysis of ceclofenc nd prcetmol reported by Gndhi et l. (14) nd spectrofluorimetric determintion of etodolc nd ceclofenc (7). British Phrmcopoei 2010 described potentiometric titrtion for the determintion of ceclofenc. In this method, 0.3 g of the drug ws dissolved in 40 ml of methnol. Then, the solution ws titrted with 0.1 M NOH with end point determined potentiometriclly. A mjority of these methods suffers from the disdvntge of extensive extrction procedures, long nlyticl time, high clibrtion rnge nd low sensitivity. In continution of previous efforts in the nlysis of orgnic compounds of phrmceuticl importnce through zo dye chemicl derivtiztion with 4-crboxy-2,6-dinitrobenzene dizonium ion (15ñ23), we report in this pper sensitive, cost effective nd rpid method for the ssy of ceclofenc in tblets. EXPERIMENTAL Chemicls nd regents Aceclofenc reference substnce, cetone, glcil cetic cid, ethyl cette, methnol, Zerodol Æ 100 mg/ tblet (Ipc Lbortories Ltd.), Oetco Æ 100 mg/tblet (Lborte Phrmceuticl Ltd.). Equipment Electrochemicl melting points pprtus (Sturt, Englnd ), nlyticl blnce (Mettler H80),

3 Sensitive spectrophotometric determintion of ceclofenc following zo dye formtion precoted TLC luminium pltes, UV lmp 254/364mm (PW Allen nd Co., Englnd), digitl colorimeter (6051 Jenwy, UK), UVñVis spectrophotometer (Perkin Elmer Lmbd 25, UK), Vortex mixer (Griffin nd George Ltd., UK), thermostted wter bth (Lngford, UK). Methods Preprtion of stock solutions A 3.0 mg quntity of ceclofenc crystls were weighed nd dissolved in 10 ml of glcil cetic cid in volumetric flsk to give solution of M. CDNBD ws prepred in concentrted sulfuric cid with sodium nitrite s previously reported (24). Evidence of dizo coupling rection A 0.1 ml liquot of the stock solution of ceclofenc ws coupled with 0.5 ml of CDNBD by vortex mixing the rection mixture for 10 s. After 20 min, the rection tube ws trnsferred into n ice bth with 5 ml of ice-cold wter. The zo dduct formed ws extrcted into 2 ml of ethyl cette. The ethyl cette ws concentrted to 1 ml. A control contining only CDNBD ws similrly treted. The smples (dduct, CDNBD nd pure ceclofenc) were spotted on 10 5 cm TLC pre-coted luminium plte. The plte ws developed using ethyl cette nd methnol (9 : 1, v/v) s mobile phse. The developed plte ws exmined using UV lmp t 254 nd 365 nm. Optimiztion studies This ws done bsed on the method of steepest scent. The effect of temperture ws studied t four levels of 30, 50, 60 nd 70 O C with ech mintined for 5 min nd 20 min. The stock solution of ceclofenc (0.1 ml) ws coupled with CDNBD (0.5 ml). At the end of ech time, the zo dduct formed ws extrcted into 5 ml of ethyl cette nd the bsorbnce redings t λ mx of 430 nm were recorded. The procedure ws repeted with replicte smples. A 0.1 ml of ceclofenc stock solution ws dded to 0.5 ml of the regent in test tubes. The effect of vried time llowed for coupling tking plce t 30 O C between ceclofenc nd CDNBD ws studied t 0, 2, 5, 10, 15, 20, 25 nd 30 min. The zo dduct formed ws processed s described bove. The procedure ws repeted with replicte smples. Determintion of stoichiometric rtio for the formtion of zo dye Aliquots of CDNBD regent; 0, 0.2, 0.25, 0.33, 0.50, 0.67, 0.75, 0.8 nd 1.0 ml were dded into nine test tubes. Ech tube ws in turn mde up to 1.0 ml with the drug stock solution. The mixture ws vortex mixed for 10 s nd processed. The zo dduct formed in ech cse ws extrcted into 5 ml of ethyl cette. A series of blnk determintions were crried out in which the volume of the drug stock solution ws replced with glcil cetic cid. The bsorbnce ws mesured t 430 nm ginst the blnk nd vlues obtined were plotted ginst the mole frction of the regent solution. Ech determintion ws crried out in duplicte. Vlidtion studies Clibrtion curve for the dduct ws prepred using concentrtion rnge of 0ñ4.8 µg/ml from the stock of ceclofenc solution in glcil cetic cid. To seven test tubes, ech contining 0.67 ml of CDNBD regent solution, 0, 0.02, 0.03, 0.04, 0.05, 0.06 nd 0.08 ml corresponding to 0, 1.2, 1.8, 2.4, 3.0, 3.6 nd 4.8 µg/ml of ceclofenc stock solution were dded, respectively. The mixture ws vortex mixed for 10 s. After 5 min, the zo dduct ws processed nd the bsorbnce redings were recorded t 430 nm nd replicte smples were prepred. The regression line eqution nd correltion coefficient were obtined from the clibrtion curve. Accurcy nd precision re the nlyticl prmeters to be determined through recovery studies. These were determined using qudruplicte smples t three concentrtion levels of 1.2, 2.4 nd 3.6 µg/ml on three different dys. The volume of ceclofenc solution corresponding to these concentrtions were mesured i.e., 0.02, 0.04 nd 0.06 ml. Ech volume ws dded into test tubes contining 0.67 ml of CDNBD regents solution one fter the other. The rection mixture ws vortex mixed for 10 s. At the end of the coupling time, the dduct formed ws extrcted into 5 ml of ethyl cette. This ws done four times for ech volume. The bsorbnce redings were recorded t 430 nm using ethyl cette s blnk solvent. A three-dy recovery study ws done nd the ccurcy nd precision were determined from the regression eqution. Appliction to tblet nlysis Two brnds of ceclofenc tblets (100 mg) were nlyzed: Zerodol (Ipc Lbortories Ltd.) nd Oetco (Lborte Phrmceuticl Ltd.). Uniformity of weight test ws crried out on ll the brnds using 20 tblets. The ssy of ctive ingredient ws determined bsed on the new nd officil methods. For the ssy of the two brnds of ceclofenc tblets, two different nlyte stock solutions (A nd B) of the sme

4 206 SEGUN A. ADERIBIGBE et l. Figure 1: Absorption spectr of ceclofenc, CDNBD nd ceclofenc-cdnbd dduct Figure 2: Optimiztion of temperture of coupling rection Figure 3: Optimiztion of coupling rection time t 30 O C Figure 4: Determintion of stoichiometric rtio for the formtion of ceclofenc-cdnbd dduct concentrtion were prepred. Bsed on weight uniformity test, 6 mg of Zerodol Æ nd 20 mg Oetco Æ powdered tblets (ech corresponding to 3 mg of ceclofenc) were weighed nd dispersed in 10 ml of glcil cetic cid in volumetric flsk. The suspension ws left overnight for complete dissolution, fter which it ws filtered. The filtrte ws then kept in smple vil nd used s drug stock solution. From ech nlyte stock solution A nd B, 0.05 ml ws trnsferred into six tubes contining 0.67 ml CDNBD regent solution. The mixture ws vortex mixed for 10 s. At the end of 5 min, the tubes were trnsferred into n ice bth nd 5 ml of icecold distilled wter ws dded to ech test tube. The dduct formed ws extrcted into 5 ml ethyl cette nd trnsferred into vils wrpped with luminium foil. The bsorbnce redings were recorded t λ mx of 430 nm on colorimeter. For the reference potentiometric titrtion, the titrnt, sodium hydroxide ws stndrdized. Zerodol Æ powder (0.563 g) nd Oetco Æ powder (1.452 g) equivlent to 0.3 g of ceclofenc were weighed nd dissolved in 40 ml of methnol. The solution ws left for 10 min nd ws titrted with 0.1

5 Sensitive spectrophotometric determintion of ceclofenc following zo dye formtion M NOH. For every 2 ml of sodium hydroxide dded, ph ws determined. After shrp increse in ph ws observed, volume of titrnt ws reduced to 1 ml. Titrtion ws done four times for ech brnd. Vlues of ph obtined were plotted ginst volume. Then, derivtive curve of ph/ V ginst volume, V ws plotted. The titre vlue ws used in clculting weight of ceclofenc nd its % content. Figure 5: Anlyticl signl stbility t 430 nm Anlyticl signl stbility A 0.05 ml quntity of ceclofenc stock solution ws dded into ech of four test tubes contining 0.67 ml of CDNBD regent solution. After processing the rection mixture s before, the ethyl cette extrct ws kept in vils lbelled A, B, C nd D. Smples A nd B were wrpped with luminium foil, while C nd D Tble 1. Anlyticl nd vlidtion prmeters for the ssy of Aceclofenc by CDNBD method. Prmeter Vlue Mximum wvelength of bsorption (λ mx ) 430 nm Beerís lw limits, (µg/ml) 1.2 ñ 4.8 Limit of detection, (µg/ml) Limit of quntittion, (µg/ml) Molr bsorptivity (Mol -1 cm 2 ) (1.19 ± 0.09) 10 4 Sndellís sensitivity, (ng/cm 2 per bsorbnce unit) Regression eqution Slope, b % confidence intervl of slope Intercept, % confidence intervl of intercept Correltion coefficient y = bx +, where y is the bsorbnce for concentrtion x µg/ml Tble 2. Intr-dy ccurcy of the proposed CDNBD spectrophotometric method. Drug Amount tken Amount found Recovery RSD Reltive (µg/ml) (µg/ml) (%) (%) b error (%) Aceclofenc Averge of six determintions; b RSD, reltive stndrd devition Tble 3. Inter-dy ccurcy of the proposed spectrophotometric method. Drug Amount tken Amount found Recovery RSD Reltive (µg/ml) (µg/ml) (%) (%) b error (%) Aceclofenc Averge of twelve determintions; b RSD, reltive stndrd devition

6 208 SEGUN A. ADERIBIGBE et l. Scheme 1. Proposed coupling pttern between Aceclofenc nd CDNBD were unwrpped. Both groups were kept on the lbortory bench. Absorbnce redings were recorded t 30 min intervl for period of 3 h t 430 nm. Interference studies A 0.05 ml quntity of the stock solution corresponding to 3 µg/ml of ceclofenc ws spiked into tblet excipients contining 5 mg ech of lctose, strch, geltin, mgnesium sterte, tlc nd mixture of the five excipients. The procedure for coupling, extrction nd smple determintion were crried out s described erlier. Blnk determintions with CDNBD in the bsence of the drug were crried out. Sttisticl nlysis The F-test nd t-test were determined nd used to compre equivlence of the new CDNBD method with the officil potentiometric method. A p-vlue less or equl to 0.05 ws tken s significnt. RESULTS AND DISCUSSION Evidence of dizo coupling rection nd selection of nlyticl wvelength The drug produced n instnt light ornge-red colortion which becme intense with time when coupled with CDNBD regent. The TLC reveled n evidence of coupling rection with the dduct hving n R f vlue (0.63), CDNBD (0.62) nd pure ceclofenc (0.79). Further evidence on TLC for the formtion of new compound ws the fluorescence exhibited by the zo dduct t 365 nm, which ws bsent in both CDNBD nd ceclofenc. The bsorption spectrum of ceclofenc shows bsorption mximum t 277 nm. The spectrum of the dduct shows three mxim t 274 nm 346 nm nd 430 nm. The blnk regent exhibits bsorption mxim t 258 nm, 333 nm nd 423 nm. The overlid bsorption spectr of ceclofenc, the

7 Sensitive spectrophotometric determintion of ceclofenc following zo dye formtion zo dduct nd CDNBD regent re presented in Figure 1. Aceclofenc hs nerly no bsorbnce t 430 nm while CDNBD hs very smll bsorbnce of bout The spectrum of the zo dduct shows bthochromic shift with respect to ceclofenc due to chromophoric elongtion cused by zo linkge. A hyperchromic shift observed in the spectrum of the dduct when compred with CDNBD ws due to higher number of chromophores. The pek t 430 nm ws selected s nlyticl wvelength, becuse t this wvelength, the difference in bsorptivity between the blnk regent nd the dduct ws mximl. Optimiztion studies The first set of optimiztion studies conducted ws the determintion of the optimum temperture nd time required for the dizo coupling rection between ceclofenc nd CDNBD. The temperture profile is presented in Figure 2. There ws n increse in bsorbnce from 30 to 50 O C t 5 min incubtion period. After 50 O C, bsorbnce decresed. At 20 min of incubtion period, bsorbnce decresed slightly from 30 to 50 O C. After 50 O C, there ws shrp decrese in bsorbnce. This could hve been due to therml decomposition of the dduct formed. It hs been previously reported tht the regent, CDNBD is resonbly stble t elevted tempertures (24). However, the drstic reduction in the bsorbnce with n increse in temperture cn be ttributed to therml decomposition of the zo dduct. Since gin in time of nlysis will be chieved on using the most convenient temperture, 30 O C ws selected s the temperture for the rection of ceclofenc with CDNBD. Further investigtion of the rection time ws crried out t 30 O C. It ws observed, s shown in Figure 3, tht the bsorbnce obtined t 0 nd 2 min ws the sme. Absorbnce rose for 5 min nd then decresed stedily until 15 min fter which it ws constnt. This mens tht coupling rection is very fst nd completed fter vortex mixing of the drug/regent mixture for 10 s. The optiml rtio for the formtion of the zo dduct ws investigted using Jobís method of continuous vrition. The result is presented in Figure 4. The highest possible bsorbnce reding ws obtined t mole frction of 0.67 for the regent solution nd the bsorbnce ws found to decrese t lower or higher mole frctions. This shows tht CDNBD nd ceclofenc rect in 2:1 stoichiometric rtio. Aceclofenc hs two romtic rings joined by n mino bridge. Both rings re fvorbly ttcked by the strong electrophile, CDNBD. The mino group (-NH) of the ceclofenc direct the electrophile to the pr position of the two romtic rings s shown in Scheme 1. This justifies the 2:1 regent to drug stoichiometric rtio obtined. TLC nlyses of the coupling rection mixture reveled the presence of single dduct thereby ruling out formtion of multiple products. The isolted nture of the romtic rings might hve promoted the simultneous romtic electrophilic substitution. The proposed rection pttern between ceclofenc nd CDNBD is presented in Scheme 1. However, further evidence suggesting the probbility of the dduct proposed comes from the rection of indomethcin with CDNBD, where 1 H NMR reveled disubstitution by CDNBD once gin due to the isolted nture of the romtic skeletons in indomethcin (18). Vlidtion studies Clibrtion lines were prepred on three successive dys nd the verge bsorbnce redings were used to describe liner regression line for the ssy of ceclofenc by the CDNBD method. The ssys of ceclofenc were liner over the rnge 1.2ñ4.8 µg/ml of ceclofenc with liner regression of y = x with correltion coefficient of The limits of detection (LOD) nd quntittion (LOQ) were estimted s stipulted by the ICH guidelines (25) using the expressions below: 3.3σ 10σ LOD = ññññññ nd LOQ = ññññññ s s where σ is the stndrd devition of the blnk signls nd s is the slope of the clibrtion grph. The LOD nd LOQ vlues obtined re nd µg/ml, respectively. The Sndellís sensitivity obtined is ng/cm 2. These low vlues point to the high sensitivity relted to this new spectrophotometric method. This is the first report of such high sensitivity for the estimtion of ceclofenc. The vrious nlyticl nd vlidtion prmeters re presented in Tble 1. Accurcy nd precision were estimted over three dys by recovery studies. The intr- nd interdy dt obtined re presented in Tbles 2 nd 3, respectively. The intermedite precision is presented in Tble 4. This represented the precision of the nlyticl method over the three ssessment periods. The ccurcy of the new spectrophotometric method using CDNBD rnged from 0.8 to 5.8% (reltive error). Lower reltive errors were obtined with higher concentrtion rnges thn the low concentrtion vlues. The overll recoveries of ceclofenc over three-dy ssessment ws found to be ± 1.85 (RSD 1.784%).

8 210 SEGUN A. ADERIBIGBE et l. Tble 4. Assessment of intermedite precision. Recovery (% ± S.D.) Drug Dy 1 Dy 2 Dy 3 Aceclofenc ± ± ± 1.37 vlues re the men of three determintions ± S.D.; overll recovery: ± 1.85 (RSD = 1.78 %) Tble 5. Men recoveries of ceclofenc in the presence of common tblet excipients. Excipients Strch Mgnesium Geltin Lctose Tlc Mixture of sterte excipients Recovery (% ± S.D.) ± 0.57 ± 0.57 ± 0.62 ± 0.57 ± 0.57 ± 0.57 n = 4 Tble 6. Assy of ceclofenc in tblets using the CDNBD nd officil potentiometric methods. Drug Amount Tblet Found Men % ± S. D. Sttistics (Amount on RSD (%) brnds (mg) lbel, mg) (New method) New method Officil method F-test t-test Aceclofenc Zerodol Æ ± ± (100 mg) Oetco Æ ± ± Averge of six determintions Interference studies The influence of commonly utilized excipients on the recovery of ceclofenc from the rection medium ws studied using strch, mgnesium sterte, geltin, lctose, tlc nd mixture of the five excipients. The results obtined re presented in Tble 5. Good recoveries were obtined in the presence of ll the excipients implying tht the method is free from undue interference by the commonly utilized tblet excipients. Anlyticl signl stbility The bsorbnce redings of the ceclofenc- CDNBD zo dduct ws monitored over three hours nd bsorbnce vlues were recorded t 30 min intervls for sets of smples wrpped nd those exposed to diffuse light of the lbortory. The result is presented in Figure 5. There is no pprecible chnge in bsorbnce for wrpped smple extrct from 0 to 60 min fter which there is mrked decline up to 180 min. The unwrpped smple extrct shows slight chnge in bsorbnce from 0 to 30 min, therefter, there is gret decline up to 180 min. The greter loss in bsorbnce by the unwrpped smples might be due to the greter light intensity pssing through the solution, compred with the wrpped smples, which might probbly llow the pssge of light vi the plstic cover of the plstic vil. From the result, it cn be inferred tht the zo dduct is mximlly stble for 1 h when wrpped. The significnt loss of bsorptivity fter this time implies tht this new method shres the common disdvntge of most color-forming derivtiztion procedures, where stbility of the colored complex or solution to diffuse light is low. Thus, it is recommended tht smple solutions of ceclofenc dduct re wrpped nd determined within 1 h fter preprtion. Tblet nlysis The new spectrophotometric method developed using romtic ring derivtiztion with CDNBD ws pplied to the ssy of ceclofenc in two tblet brnds nd the results were compred with potentiometric titrtion using NOH (Tble 6). No significnt difference ws found in the content of ceclofenc in the tblets by both methods thereby estblishing their equivlence. Some clerly defined dvntges of this new spectrophotometric method re: low concentrtions of the clibrtion rnges compred to the use of Folin-Cioclteu nd p-dimethylminocinnmlde-

9 Sensitive spectrophotometric determintion of ceclofenc following zo dye formtion hyde s derivtizing gents. Singhvi nd Goyl (8) nd El-Moghzy et l. (6) reported rnges of 80ñ160 µg/ml nd 20ñ100 µg/ml, respectively. These two Beerís limits re higher thn tht of CDNBD method (1.2ñ4.8 µg/ml) conferring gret dvntge of sensitivity to the ltter. Furthermore, CDNBD method is simple, fst nd rpid with n instnt formtion of stble orngered dduct compred with the procedure reported (6), where stble complex is formed fter heting t 75 O C for 20 min. In ddition, dilution of stock solution with phosphte buffer ph 7.4 nd distilled wter to obtin working solution s reported by Shh et l. (9) my compromise ccurcy nd precision. The CDNBD method is the first described method for the nlysis of ceclofenc by zo dye derivtiztion methodology. CONCLUSIONS The new spectrophotometric method described in this report is simple, ccurte, precise nd highly sensitive compred to previously reported methods. It is of equivlent ccurcy with the reference potentiometric titrtion. The method cn find ppliction s simple nd rpid in-process qulity control procedure for the ssy of ceclofenc. REFERENCES 1. British Phrmcopoei Commission: British Phrmcopoei 2010, Vol. 1, p. 18. The Phrmceuticl Press, London The Merck Index 12 th edn, p. 21, Merck Reserch Lb, division of Merck nd Co. Whitehouse Sttion, N.J Ymzki R., Kwi S., Mtsuzki T., Hshimoto S., Yokohum T., Mizushim Y.: Eur. J. Phrmcol. 329, 181 (1997). 4. Mneiro E., Lopez-Armnd M.J., Fernndez- Sueiro J.L., Lem B., Gldo F., Blnco F.J.: J. Rheumtol. 28, 2692 (2001). 5. Mrtindle: The Complete Drug Reference. 36th edn., p. 14; The Phrmceuticl Press, London El-Moghzy A.S.M., Zwill N.H., Mohmmd A.A., El-Kousy N.M.: J. Phrm. Biomed. Anl. 27, 243 (2002). 7. El- Kousy N.M.: J. Phrm. Biomed. Anl. 20, 185 (1999). 8. Singhvi I., Goyl A.: Ind. J. Phrm. Sci. 69, 164 (2007). 9. Shh R., Mgdum C., Kumr S., Kumr D., Nikwde N., Res. J. Phrm. Technol. 1, 430 (2008). 10. Hsn N.Y., Abdel-Elkwy M., Elzeny B.F., Wgieh N.E.: Frmco 58, 91 (2003). 11. El-Shrty Y.S., Reft M., El-Khteeb S.Z.: Drug Dev. Ind. Phrm.: 28, 571 (2002). 12. Pwr U.D., Nik A.V., Sulebhvikr A.V., Dtr T.A., Mngonkr K.V.: E-J. Chem. 6, 289 (2009). 13. Vidy V.V., Singh G.R., Choukekr M.P., Kekre M.B.: E-J. Chem. 7, 260 (2010). 14. Gndhi S.V., Brhte N.S., Ptel B.R., Pnchl D.D., Bothr K.G.:Act Chromtogr. 20, 175 (2008). 15. Idowu S.O., Tmbo S.C., Adegoke A.O., Olniyi A.A. : Trop. J. Phrm. Res. 1, 15 (2002). 16. Idowu S.O., Adegoke A.O., Olniyi A.A. : J. AOAC Int. 87, 573 (2004). 17. Idowu S.O., Adegoke A.O., Oderinu B.A., Olniyi A.A.: Pk. J. Phrm. Sci. 19, 134 (2006). 18. Adegoke O.A., Idowu S.O., Olniyi A.A.: Act Phrm. 56, 189 (2006). 19. Adegoke O.A., Idowu S.O., Olniyi A.A.: J. Irn. Chem. Soc. 3, 277 (2006). 20. Adegoke O.A., Idowu S.O., Olniyi A.A.: Trop. J. Phrm. Res. 6, 695 (2007). 21. Adegoke O.A., Idowu S.O., Olniyi A.A.: Afr. J. Med. Med. Sci. 36, 249 (2007). 22. Idowu S.O., Adegoke O.A., Adeniji A.O., Olniyi A.A.: Est Cent. Afr. J. Phrm. Sci. 12, 8 (2009). 23. Adegoke O.A., Idowu S.O., Drmol O.P., Ogunsny O.S.: Act Phrm. Sci. 52, 269 (2010). 24. Idowu O.S., Kolwole A.O., Adegoke O.A., Kolde Y.T., Fsnmde A.A., Olniyi A.A.: J. AOAC Int. 88, 1108 (2005). 25. ICH Topic Q2 (R1), Vlidtion of Anlyticl Procedures: Text nd Methodology (CPMP/ ICH/281/95); ccessed June Received: