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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490ISSN:2249-5347IJSIDInternational Journal of Science Innovations and DiscoveriesAn International peerReview Journal for ScienceResearch ArticleAvailable online through www.ijsidonline.infoSINGLE RP-HPLC METHOD FOR THE QUANTIFICATION OF ACECLOFENAC, PARACETAMOL ANDCHLOROZOXAZONE IN FORMULATIONSHari kishan Reddy Ganthi 1* , Hanimi Reddy Bapatu 2 , Maram Ravi Kumar 3 , Useni Reddy Mallu 1 ,1Dept. of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India; 2 Department of Chemistry,JNT University, Kukatpally, Hyderabad, AP, India-500072.; 3 AR&D, Custom Pharmaceutical Services,Dr. Reddy’s Laboratories Ltd, Bachupally, Hyd-72, India.Received: 14-08-2012Accepted: 17-10-2012ABSTRACT*Corresponding AuthorAddress:Name:Hari Kishan Reddy GanthiPlace:Sri Krishnadevaraya UniversityAnantapur, AP, IndiaE-mail:kishangan05ster@gmail.comObjectives: To develop a single RP-HPLC method for determination of Aceclofenac,Paracetamol and Chlorozoxazone contents in formulation.Methods: Chromatographic separation was achieved on Inertsil ODS 3V, 150 x4.6mm, 5µcolumn. Mobile phase composed of phosphate b u f f e r o f p H 6 . 0 a n da c e t o n i t r i l e i n t h e r a t i o n 6 7 : 3 3 v / v . 1.0ml per min flow rate and detectionwas at 275 nm.Results: High resolution was achieved with the simple mobile phase composition andretention time of Paracetamol, Chlorozoxazone, and Aceclofenac are about 2.1min, 8.8minand 20.7min, respectively. The area of all ingredient peaks were a linear function ofconcentration in the range 150.4 to 752.3 ppm for Paracetamol, 120.2 to 761.4 ppm forChlorozoxazone and 29.9 to 159.9 ppm for Aceclofenac and the correlation co-efficientINTRODUCTIONvalue of all active ingredients within the limit (0.999).Conclusion: Proposed HPLC method was validated with specificity, linearity, accuracy,reproducibility and ruggedness and it is applicable for regular analysis.Keywords: Aceclofenac, Paracetamol, Chlorozoxazone and RP-HPLC method.INTRODUCTIONInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012471

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490INTRODUCTIONParacetamol (acetaminophen) is one of the most popular over-the-counter (OTC) analgesic and antipyretic drugs.Paracetamol (1-8) is available in different dosage forms: tablet, capsules, drops, elixirs, suspensions and suppositories.Aceclofenac is a non-steroidal anti-inflammatory drug (NSAID). It is used for the relief of pain and inflammation inrheumatoid arthritis, osteoarthritis and ankylosing spondylitis. The dose is 100 mg twice daily. It should not be given topeople with porphyria or breast-feeding mothers, and is not recommended for children.Chlorzoxazone is used to relieve pain and stiffness caused by muscle strains and sprains. It is used in combination withphysical therapy, analgesics (such as aspirin or acetaminophen), and rest. The side effects are upset stomach, drowsiness,dizziness, lightheadedness, weakness, skin rash or itching, yellowing of the skin or eyes and stomach pain.Chemical structures of all ingredients were represented in figure-1.All three ingredients are available in liquid pharmaceuticaldosage forms.Paracetamol Chlorozoxazone AceclofenacFigure-1: Chemical structure of all ingredientsAll ingredients have reported methods for individual and other combination products, but the objective of the presentstudy is to develop a single RP-HPLC method for the estimation of Paracetamol, Aceclofenac and Chlorozoxazone informulations and the developed and validated method is simple, novel, rugged and linear.MATERIALS AND METHODSSelection of mobile phase:Various buffer salts, pH values were tried with different organic solvents (acetonitrile or methanol) for the optimization ofmobile phase. Finally well shaped and high resolution was achieved with pH 6.0 phosphate buffer and acetonitrile at 67:33 v/vratio.Chemicals and reagents:Ortho phosphoric acid, triethyl amine (AR Grade) was procured from S.D fine chemicals. High pure (NLT 98.5%) standards(Aceclofenac, Paracetamol and Chlorozoxazone) were used for this study. HPLC grade acetonitrile were procured fromSpectrochem Pvt. Ltd. Water is prepared by mili Q system (Milli-pore).Buffer preparation: Diluted 2.0mL of ortho phosphoric acid to 1000 mL of water. Adjusted to pH 6.0 with triethyl amineFiltered through 0.45µm membrane filter and degassed.Mobile Phase: Mixed the buffer and acetonitrile in the ration 67:33 v/v.Diluent: Mobile phase.HPLC conditions:Column : Inertsil ODS 3V (150X4.6mm, 5μm)Wavelength : 275nmInjection volume:20 μLInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012472

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Flow rate : 1.0 mL/minTemperature : 30°CRun time : 30minMobile phase : Buffer and Acetonitrile 67:33 v/vPreparation of standard solution: For 100/ 500 / 500 mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration ofAceclofenac is about 2000µg/mL)Accurately weigh and transfer 100 mg of Paracetamol working standard, and 100 mg of Chlorzoxazone working standardin to 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute upto the mark with mobile phase and mix well. (Concentration of Aceclofenac is about 100 µg/mL, concentration ofParacetamol is about 500µg/mL and concentration of Chlorzoxazone is about 500µg/mL ).Preparation of standard solution: For 100/ 325/ 250 mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration ofAceclofenac is about 2000µg/mL)Accurately weigh and transfer 65 mg of Paracetamol working standard, and 50 mg of Chlorzoxazone working standard into 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up tothe mark with mobile phase and mix well. (Concentration of Aceclofenac is about 100 µg/mL, concentration ofParacetamol is about 325µg/mL and concentration of Chlorzoxazone is about 250µg/mL).Preparation of standard solution: For 100/ 500 mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration ofAceclofenac is about 2000µg/mL)Accurately weigh and transfer 100 mg of Paracetamol working standard, in to 200 mL volumetric flask, add to it about 10mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to the mark with mobile phase and mix well.(Concentration of Aceclofenac is about 100 µg/mL, concentration of Paracetamol is about 500µg/mL).Preparation of standard solution: For 100/ 500/15 mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration ofAceclofenac is about 2000µg/mL)Accurately weigh and transfer 100 mg of Paracetamol working standard, in to 200 mL volumetric flask, add to it about 10mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to the mark with mobile phase and mix well.(Concentration of Aceclofenac is about 100 µg/mL, concentration of Paracetamol is about 500µg/mL).Preparation of standard solution: For 100mg Tablets:Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50.0 mL volumetric flask, add to itabout 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentrationof Aceclofenac is about 2000µg/mL)International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012473

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Dilute the 10 mL of above Aceclofenac stock solution to 200 mL with mobile phase and mix well. (Concentration ofAceclofenac is about 100 µg/mL).Preparation of test solution: For 100/ 500 / 500 mg Tablets:Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weighaccurately the powder equivalent to 500 mg of Paracetamol into a 200 mL volumetric flask, add to it 20 ml ofacetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minuteswith intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix.Filter the solution through 0.45µ nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL withdiluent.Preparation of test solution: For 100/ 325 / 250 mg Tablets:Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weighaccurately the powder equivalent to 325 mg of Paracetamol into a 200 mL volumetric flask, add to it 20 ml ofacetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minuteswith intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix.Filter the solution through 0.45µ nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL withdiluent.Preparation of test solution: For 100/ 500 / 15 mg Tablets:Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weighaccurately the powder equivalent to 500 mg of Paracetamol into a 200 mL volumetric flask, add to it 20 ml ofacetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minuteswith intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix.Filter the solution through 0.45µ nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL withdiluent.Prepare the test solutions in duplicate.Procedure:Equilibrate the column with mobile phase for sufficient time until stable baseline is obtained. Inject blank, standard andsample preparation filter through 0.45µ nylon filter. Inject blank (diluent) in single, standard preparation in fivereplicates, and each test preparation into the chromatographic system and record the chromatograms. Inject standardpreparation as a bracketing after every six injections of test preparations. Evaluate the system suitability parameters fromthe standard chromatograms. The order of elution is Paracetamol, Chlorzoxazone and Aceclofenac.Calculation: % content= Test solution area x Std. Dilution factor x Std. PotencyStandard solution area x Test dilution factorRESULTS AND DISCUSSIONSystem suitability:Standard solution was prepared as per the proposed test method and injected into the HPLC system. The results of thesystem suitability assessment for initial validation study parameters were tabulated in Table 1.International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012474

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Sr.No.1System Suitability parameterThe % RSD of peak area response forfive replicate injections of standardTable 1: System suitabilityObservationsLimitsAceclofenac Paracetamol Chlorzoxazone0.3 0.1 0.2 NMT 2.02 Theoretical plates 2963 1506 3986 NLT 10003 Tailing factor 2.0 1.8 1.9 NMT 2.0Precision Studies:System Precision:Standard solution of working standard was prepared as per the proposed test procedure for repeatability studies. Fivereplicate injections were injected into the HPLC system. % RSD for the peak responses as the peak area was calculated,Results are tabulated in Table 2.Table 2: System PrecisionInjection No.Area ResponseAceclofenac Paracetamol Chlorzoxazone1 3481243 9580728 141479182 3480070 9596079 141922753 3474685 9605600 142006134 3456683 9587599 141480735 3462315 9597724 14154756Average 3470999 9593546 14168727SD 10967.78 9602.586 25622.336% RSD 0.3 0.1 0.2Method Precision:Six test preparations were prepared as per the proposed test method for individual test preparation. All individual testpreparations were injected into the HPLC system as per the test method. The %assay results were calculated for eachindividual test sample, along with average assay and % RSD for the six preparations. The results are tabulated in Table 3.Table 3: Method Precision StudiesSr. No% AssayAceclofenac Paracetamol Chlorzoxazone1 98.4 95.8 103.42 98.1 95.9 103.43 98.7 95.8 103.54 98.6 96.1 103.95 98.2 95.9 103.56 98.8 95.6 103.2Average 98.5 95.9 103.5SD 0.2805 0.1643 0.2317% RSD 0.3 0.2 0.2Ruggedness (Intermediate precision):Six test preparations were prepared as per the proposed test method for individual test preparation and injected into thedifferent HPLC system by the different analyst using different make of HPLC Column at different day.The %assay results were calculated for each of the sample for each of the variability. Average assay values and % RSD forthe six preparations were calculated. The results are tabulated in Table 4.International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012475

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Table 4: RuggednessSr. No% AssayAceclofenac Paracetamol Chlorzoxazone1 98.8 95.8 103.82 98.4 95.7 103.73 98.6 95.8 103.84 98.8 96.1 104.05 98.9 96.0 103.96 98.9 96.0 103.9Average 98.7 95.9 103.9SD 0.1966 0.1549 0.1049% RSD 0.2 0.2 0.1Table 5: Ruggedness data evaluationSr. No% AssayAceclofenac Paracetamol Chlorzoxazone1 98.4 98.8 95.8 95.8 103.4 103.82 98.1 98.4 95.9 95.7 103.4 103.73 98.7 98.6 95.8 95.8 103.5 103.84 98.6 98.8 96.1 96.1 103.9 104.05 98.2 98.9 95.9 96.0 103.5 103.96 98.8 98.9 95.6 96.0 103.2 103.9OverallAverage98.6 95.9 103.7Overall SD 0.2697 0.1545 0.2570Overall% RSD0.3 0.2 0.2Linearity and Range:Linearity of Detector Response:The linearity studies of detector response for analytes were evaluated in the concentration range from about 50% of lowerstrength to 150% of the higher strength of the targeted concentration. The diluted standard solutions were prepared fromstock solution in the above range and analysed using proposed analytical method by injecting each level in to the system.The Linearity graph of average area response verses concentration was plotted and the correlation coefficient wascalculated. The results are tabulated in Table 6.Table6: Linearity of Detector Response:Aceclofenac Paracetamol ChlorzoxazoneSr. No Concentration(g/mL)ArearesponseConcentration(g/mL)ArearesponseConcentration(g/mL)Arearesponse1 29.9976 1051778 150.4635 2644943 120.2281 36036322 59.9951 2105682 200.6180 3519271 200.3802 60477723 75.9938 2451169 401.2361 7010633 320.6083 96117414 99.9919 3503313 501.5451 8745948 500.9505 149467985 109.9911 3849570 551.6996 9595498 601.1406 178849436 119.9903 4187013 626.9314 10901381 721.3687 213237307 159.9870 5600698 752.3177 13044334 761.4448 22494050CorrelationCoefficient0.999 1.000 1.000Slope 35288.4907 17291.5590 29427.4152Intercept 56878.5049 55950.5535 139465.4305International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012476

Linearity of Test Method (Inferred from Accuracy):Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490The results obtained in accuracy study were further interpreted for the evaluation of linearity of the test method. Theresults of average of mg added at each spiked level was plotted against average mg recovered at each accuracy level and thecorrelation coefficient for set of data was evaluated. The results are tabulated in Table 7.For 100/500/500 mg Tablets:Recovery levelAmountAdded (mg)Table 7: Linearity of Test method (Inferred from accuracy)Aceclofenac Paracetomol ChlorzoxazoneAmountRecovered(mg)AmountAdded(mg)AmountRecovered(mg)AmountAdded(mg)AmountRecovered(mg)80% 79.4 81.0 397.2 401.0 397.1 403.0100% 99.6 101.0 495.8 498.0 495.6 502.0120% 119.6 121.0 594.8 590.0 595.0 594.0CorrelationCoefficient1.000 0.9999 0.9997Slope 0.995 0.9565 0.9651Intercept 1.9627 21.9933 21.0867Precision at Lower and Higher extreme concentrations:Precision at lower and higher extreme range concentration for Aceclofenac (29.9976 µg/ml and 159.9870 µg/ml),Paracetamol (150.4635 µg/ml and 752.3177 µg/ml), and Chlorzoxazone (120.2281 µg/ml and 761.4448 µg/ml) ofLinearity levels were determined as per proposed method by injecting six replicate injections of standards for both thelevels. % RSD for peak responses for both the level was evaluated. The results are tabulated in Table 8.Table 8: Precision at Lower and Higher extreme Levels of linearityArea ResponsesArea Responses Aceclofenac Area Responses ParacetamolChlorzoxazoneInjection NumberLower level Higher level Lower level Higher level Lower level Higher level(25%) (150%) (25%) (150%) (25%) (150%)1 1051198 5593151 2654481 13053921 3607531 224874582 1053062 5599304 2655088 13029744 3605991 224757483 1052477 5593984 2646499 13073632 3602871 225549934 1051003 5598529 2637079 13028568 3600717 224720925 1052376 5602959 2635339 13056944 3598395 225061956 1050552 5616261 2641174 13023193 3606288 22467814Average 1051778 5600698 2644943 13044334 3603632 22494050SD 993.4749 8438.0317 8539.324 20088.1667 3586.7515 32895.1346% RSD 0.1 0.2 0.3 0.2 0.1 0.1Accuracy:An accuracy study was conducted by spiking the known amount of analytes in the equivalent weight of placebo. Accuracystudy was conducted in triplicate at three different levels, (80%, 100% and 120% of target concentration). The sampleswere analyzed as per the proposed test procedure and the % recovery for each spike level was calculated. The precision ateach spike level was also established.The results are tabulated in Table 9, 10 and 11.International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012477

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Table 9: Accuracy of AceclofenacRecoveryLevel80%100%120%RecoveryLevel80%100%120%AceclofenacAmountAmountAdded (mg) Recovered (mg)% Recovery79.4 80.5 101.479.4 80.9 101.979.5 80.4 101.199.6 101.7 102.199.6 101.1 101.599.6 101.2 101.6119.6 119.8 100.2119.6 121.3 101.4119.6 122.4 102.3AverageRecovery (%)% RSD101.5 0.4101.7 0.3101.3 1.0Overall 101.5 0.6Table 10: Accuracy ParacetamolParacetamolAmountAmountAdded (mg) Recovered (mg)% Recovery397.1 399.9 100.7397.1 402.6 101.4397.3 400.3 100.8495.8 500.1 100.9495.8 495.9 100.0495.9 496.5 100.1594.8 582.6 97.9594.8 590.9 99.3594.8 597.3 100.4AverageRecovery (%)% RSD101.0 0.4100.3 0.599.2 1.3Overall 100.2 1.0Table 11: Accuracy of ChlorzoxazoneRobustness:RecoveryLevel80%100%120%ChlorzoxazoneAmountAmountAdded (mg) Recovered (mg)% Recovery397.1 403.6 101.6397.1 405.6 102.1397.2 401.1 101.0495.6 502.6 101.4495.7 502.0 101.3495.6 502.4 101.4595.0 588.2 98.9595.0 591.4 99.4595.0 601.3 101.1Effect of variation in Flow rate of mobile phase (±10%):AverageRecovery (%)% RSD101.6 0.5101.4 0.199.8 1.2Overall 100.9 1.0Mobile phase Flow rate as per proposed analytical method is 1.0 ml/min. Change in flow rate by –10% = 0.9 ml/min. Change inflow rate by +10% = 1.1 ml/min. The effect due to change in flow rate on the system suitability parameters are compared.Results are tabulated in Table 12.International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012478

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Table 12: System suitability of change in Flow RateSr.ObservationsSystem Suitability parameterNo.As Such - 10% + 10%The % RSD of Aceclofenac 0.1 0.1 0.2peak area Paracetamol 0.1 0.1 0.21 response for fivereplicateChlorzoxazone 0.1 0.2 0.3injectionsAceclofenac 6209 6301 61582 Theoretical plates Paracetamol 2559 2697 2413Chlorzoxazone 10476 10660 9988Aceclofenac 2.0 2.0 2.03 Tailing factor Paracetamol 1.4 1.4 1.3Chlorzoxazone 1.1 1.1 1.1Effect of variation in Column oven temperature (±5°C):LimitsNMT 2.0NLT 1000NMT 2.0The Column Oven temperature as per proposed analytical method is 30°C. Change in Column oven Temperature by –5°C =25°C. Change in Column oven Temperature by +5°C = 35°C. The effect due to change in Column oven temperature on thesystem suitability parameters are compared. Results are tabulated in Table 13.Sr.No.Table 13: System suitability of change in Column Oven temperatureSystem Suitability parameterObservationsAs Such -5°C + 5°C1The % RSD of peakarea response for fivereplicate injectionsAceclofenacParacetamolChlorzoxazone0.10.10.10.10.00.00.50.10.1Aceclofenac 6209 5881 65312 Theoretical plates Paracetamol 2559 2434 2497Chlorzoxazone 10476 9784 9947Aceclofenac 2.0 2.0 2.03 Tailing factorParacetamol 1.4 1.4 1.4Chlorzoxazone 1.1 1.1 1.1Effect of variation in organic phase composition (± 10%):LimitsNMT 2.0NLT 1000NMT 2.0The Organic phase ratio of mobile phase as per proposed analytical method is 0.2% v/v Orthophosphoric acid: Acetonitrile is67:33 v/v. Change in Organic phase ratio of mobile phase by +10% and -10% of 0.2% v/v Orthophosphoric acid:Acetonitrile was performed. The effect due to change in organic phase composition on the system suitability parameters arecompared. Results are tabulated in Table 14.Table 14: System suitability of change in organic phase compositionSr.ObservationsSystem Suitability parameterNo.As Such -5% + 5%The % RSD of peak Aceclofenac 0.1 0.2 0.11 area response for five Paracetamol 0.2 0.1 0.2replicate injections Chlorzoxazone 0.4 0.1 0.1Aceclofenac 5973 6363 51762 Theoretical plates Paracetamol 2526 2216 2581Chlorzoxazone 8420 8537 7227Aceclofenac 1.6 1.3 1.43 Tailing factorParacetamol 1.4 1.3 1.4Chlorzoxazone 1.1 1.1 1.1LimitsNMT 2.0NLT 1000NMT 2.0International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012479

Prep. No.Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Table 19: Filter paper interference study ChlorzoxazoneChlorzoxazoneAverage Standard AreaSTD-1 % Diff STD-2%DiffAverage Test AreaTest-1 % Diff Test-2Unfiltered 14171478 NA 14113560 NA 15017843 NA 15040877 NA0.45µFilter 14143527 0.2 14114024 0.0 14979503 0.3 14994880 0.3%DiffPeak Purity results of Sample As such:Test preparation was prepared as per the test method and injected into HPLC system. The peak purity result wasevaluated by Photo Diode Array Detector.The results are tabulated in Table 20 and 21.Table 20: Sample As such Higher Strength 100/500/500 mg tabletsPeak purity resultsParameter Aceclofenac Chlorzoxazone ParacetamolPurity-1 angle 0.071 0.271 2.092Purity-1 threshold 0.251 0.282 6.619Purity Flag No No NoTable 21: Sample As such Lower Strength 100/500/15 mg tabletsPeak purity resultsParameter Aceclofenac ParacetamolPurity-1 angle 0.057 2.171Purity-1 threshold 0.232 6.775Purity Flag No NoForced Degradation studies:The stress degradation study was carried out on the sample preparations (100/500/500 mg and 100/500/15 mg strength)of Tablet, and the degradation was evaluated by calculating the % degradation of in comparison with unstressed samplepreparation. The degradation of 10-30% was tried by following stress conditions to prove the stability indicatingcharacteristics of the method. The stress conditions and results were compiled in Table 22 and 23.Stress ConditionAcid degradation0.1N HCl, 1hr at 60°CAlkali degradation1N NaOH, 1hr at 80°CPeroxide degradation6% H 2O 2, 2hrs at 80°CThermal degradation2 hrs at 80°CPhotolytic degradation1.2 m.lux hrsTable 22: % degradation data for 100/500/500 mg tabletsAceclofenac Paracetamol Chlorzoxazone%%%degradationdegradationdegradation16.2 0.1 3.295.1 9.9 7.017.5 0.5 1.911.3 1.4 1.412.0 1.4 1.6International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012481

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Table 23: % degradation data for 100/500/15 mg tabletsAceclofenacParacetamolStress Condition%degradation%degradationAcid degradation 0.1N HCl, 1hr at 60°C 10.7 3.6Alkali degradation 1N NaOH, 1hr at 80°C 98.6 19.2Peroxide degradation 6% H 2O 2, 2hrs at 80°C 17.4 2.5Thermal degradation 2 hrs at 80°C 8.2 3.0Photolytic degradation 1.2 m.lux hrs 8.2 3.0Solution Stability Studies:Mobile phase stability at room temperature:The mobile phase was prepared as per the proposed test method and kept at room temperature for a period of two days inwell closed condition. The system suitability solutions were prepared and injected into the HPLC system at initially andperiodically after 1day. The system suitability parameters were evaluated. The mobile phase was also observed for Physicalchanges like haziness or precipitation. System suitability results are tabulated in Table 24.Table 24: Mobile phase stability at room temperatureSr. No.System Suitability parameterObservationsInitialDay-1The % RSD of peak area Aceclofenac 0.2 0.11 response for five replicate Paracetamol 0.2 0.2injectionsChlorzoxazone 0.1 0.1Aceclofenac 6737 67042 Theoretical platesParacetamol 3013 3227Chlorzoxazone 10870 10725Aceclofenac 1.9 1.83 Tailing factorParacetamol 1.3 1.2Chlorzoxazone 1.0 1.0Analytical Solution Stability (Standard and Test preparation) at room temperature:International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012LimitsNMT 2.0NLT 1000NMT 2.0Standard and test preparations were prepared as per the proposed test method and the stock solutions were kept at roomtemperature. The solutions were diluted freshly at each time and injected into the HPLC system at initially and at differenttime intervals. The % difference in area was calculated against the fresh standard solution injected at each time interval.The % difference in area response was evaluated against the initial assay value. The results are tabulated in Table 25, 26and 27.Table 25: Stability of Aceclofenac Standard and test solution at room temperatureAceclofenac StandardAceclofenac TestTime (Hours) Area Response % Difference Area Response % DifferenceInitial 3549534 NA 3598615 NA4 Hrs 35 min 3539237 0.3 3578216 0.68 Hrs 3540881 0.2 3609559 0.312 Hrs 15 min 3559542 0.3 3597509 0.016 Hrs 20 min 3546836 0.1 3575222 0.720 Hrs 25 min 3540089 0.3 3575272 0.623 Hrs 3557104 0.2 3571046 0.824 Hrs 30 min 3528644 0.6 3562606 1.029 Hrs 3532159 0.5 3581784 0.533 Hrs 3581358 0.9 3581157 0.540 Hrs 50 min 3630421 2.3 3581119 0.5482

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Table 26: Stability of Paracetamol Standard and test solution at room temperatureParacetamol StandardParacetamol TestTime (Hours) Area Response % Difference Area Response % DifferenceInitial 9533772 NA 9674499 NA4 Hrs 35 min 9485553 0.5 9681641 0.18 Hrs 9529119 0.0 9720005 0.512 Hrs 15 min 9562369 0.3 9713140 0.416 Hrs 20 min 9558274 0.3 9703185 0.320 Hrs 25 min 9551274 0.2 9705705 0.323 Hrs 9554327 0.2 9673281 0.024 Hrs 30 min 9551111 0.2 9659821 0.229 Hrs 9541020 0.1 9661378 0.133 Hrs 9619965 0.9 9674547 0.040 Hrs 50 min 9760962 2.4 9661793 0.1Table 27: Stability of Chlorzoxazone Standard and test solution at room temperatureChlorzoxazone StandardChlorzoxazone TestTime (Hours) Area Response % Difference Area Response % DifferenceInitial 14101688 NA 14905697 NA4 Hrs 35 min 14074515 0.2 14896823 0.18 Hrs 14105493 0.0 15018775 0.812 Hrs 15 min 14182706 0.6 14980781 0.516 Hrs 20 min 14175597 0.5 14922091 0.120 Hrs 25 min 14185354 0.6 14925356 0.123 Hrs 14165758 0.5 14885819 0.124 Hrs 30 min 14150058 0.3 14871542 0.229 Hrs 14064098 0.3 14908388 0.033 Hrs 14290784 1.3 14911371 0.040 Hrs 50 min 14499323 2.8 14930540 0.2BLANK CHROMATOGRAMInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012483

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490STANDARD CHROMATOGRAMTEST CHROMATOGRAMLINEARITY OF DETECTOR RESPONSE OF ACECLOFENACInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012484

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490LINEARITY OF DETECTOR RESPONSE OF PARACETAMOLLINEARITY OF DETECTOR RESPONSE OF CHLORZOXAZONELINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF ACECLOFENACInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012485

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF PARACETAMOLLINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF CHLORZOXAZONEAS SUCH SAMPLE CHROMATOGRAM HIGHER STRENGTHInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012486

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490AS SUCH SAMPLE PURITY PLOT HIGHER STRENGTHAS SUCH SAMPLE CHROMATOGRAM LOWER STRENGTHInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012487

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490AS SUCH SAMPLE PURITY PLOT LOWER STRENGTHACID DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTHInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012488

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490ALKALI DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTHPEROXIDE DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTHTHERMAL DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTHInternational Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012489

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490PHOTOLYTIC DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTHCONCLUSIONThe complete study results reveals that the developed and validated RP-HPLC method is accurate, precise, robust andstability indicating. This method has wide applicability and useful for regular quality control analysis of formulation samples.REFERENCES1. Granberg RA, Rasmuson AC, Solubility of paracetamol in pure solvents, Journal of Chemical & Engineering Data, 1999, 44(6), 1391–1395.2. Acetaminophen Drugs.com3. Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S, Paracetamol: new vistas of an old drug, CNS drug reviews,2006, 12 (3–4), 250–275.4. Altinoz MA, Korkmaz R, NF-kappaB, macrophage migration inhibitory factor and cyclooxygenase-inhibitions as likelymechanisms behind the acetaminophen- and NSAID-prevention of the ovarian cancer, Neoplasma, 2004, 51 (4), 239–247.5. Moller, P, Sindet-Pedersen S, Petersen C, Juhl G, Dillenschneider A, Skoglund L, Onset of acetaminophen analgesia:comparison of oral and intravenous routes after third molar surgery, British journal of anaesthesia, 2005, 94 (5): 642–648.6. Viswanathan, Feskanich, Schernhammer ES, Aspirin, NSAID and Acetaminophen use and the Risk of Endometrial Cancer,Cancer Research, 2008, 68 (7), 2507.7. Acetaminophen, chemicalland21.com, 2011.8. Byrant, Bronwen, Knights, Katleen, Salerno, Evelyn, Pharmacology for health professionals, Elsevier, 2007, 270.9. International Conference on Harmonization, Q2A: Text on Validation of Analytical Procedures, Federal Register, 1995, 60(40), 11260–11262.10. FDA, Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation, Availability,Federal Register (Notices), 2000, 65(169), 52776–52777.11. International Conference on Harmonization, Q2B: Validation of Analytical Procedures: Methodology andAvailability, Federal Register, 1997, 62 (96), 27463–27467.12. USP 25–NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal Convention, Rockville,Maryland, USA, 2002), 2256.International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012490