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Single rp hplc method for the quantification of aceclofenac, paracetamol and chlorozoxazone in formulations
 

Single rp hplc method for the quantification of aceclofenac, paracetamol and chlorozoxazone in formulations

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    Single rp hplc method for the quantification of aceclofenac, paracetamol and chlorozoxazone in formulations Single rp hplc method for the quantification of aceclofenac, paracetamol and chlorozoxazone in formulations Document Transcript

    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 ISSN:2249-5347 IJSID International Journal of Science Innovations and Discoveries An International peer Review Journal for Science Research Article Available online through www.ijsidonline.info SINGLE RP-HPLC METHOD FOR THE QUANTIFICATION OF ACECLOFENAC, PARACETAMOL AND CHLOROZOXAZONE IN FORMULATIONS 1Dept. of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India; 2Department of Chemistry, Hari kishan Reddy Ganthi1*, Hanimi Reddy Bapatu2, Maram Ravi Kumar3, Useni Reddy Mallu1, 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: 17-10-2012 ABSTRACT Objectives: To develop a single RP-HPLC method for determination of Aceclofenac, Paracetamol and Chlorozoxazone contents in formulation.*Corresponding Author Methods: Chromatographic separation was achieved on Inertsil ODS 3V, 150 x4.6mm, 5µ column. Mobile phase composed of phosphate b u f f e r of pH 6.0 and a 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 detection was at 275 nm. Results: High resolution was achieved with the simple mobile phase composition and retention time of Paracetamol, Chlorozoxazone, and Aceclofenac are about 2.1min, 8.8min and 20.7min, respectively. The area of all ingredient peaks were a linear function of concentration in the range 150.4 to 752.3 ppm for Paracetamol, 120.2 to 761.4 ppm for Chlorozoxazone and 29.9 to 159.9 ppm for Aceclofenac and the correlation co-efficientAddress: value of all activeINTRODUCTION limit (0.999). ingredients within theName: Conclusion: Proposed HPLC method was validated with specificity, linearity, accuracy,Hari Kishan Reddy GanthiPlace: reproducibility and ruggedness and it is applicable for regular analysis.Sri Krishnadevaraya University Keywords: Aceclofenac, Paracetamol, Chlorozoxazone and RP-HPLC method.Anantapur, AP, IndiaE-mail:kishangan05ster@gmail.com INTRODUCTION 471 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490Paracetamol (acetaminophen) is one of the most popular over-the-counter (OTC) analgesic and antipyretic drugs. INTRODUCTIONParacetamol (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 Aceclofenac Figure-1: Chemical structure of all ingredients All 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 METHODSVarious buffer salts, pH values were tried with different organic solvents (acetonitrile or methanol) for the optimization ofSelection of mobile phase:mobile phase. Finally well shaped and high resolution was achieved with pH 6.0 phosphate buffer and acetonitrile at 67:33 v/vratio.Ortho phosphoric acid, triethyl amine (AR Grade) was procured from S.D fine chemicals. High pure (NLT 98.5%) standardsChemicals and reagents:(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.Column : Inertsil ODS 3V (150X4.6mm, 5μm)HPLC conditions:Wavelength : 275nmInjection volume: 20 μL 472 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • 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/vAccurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it aboutPreparation of standard solution: For 100/ 500 / 500 mg Tablets:30 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 ).Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it aboutPreparation of standard solution: For 100/ 325/ 250 mg Tablets:30 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).Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it aboutPreparation of standard solution: For 100/ 500 mg Tablets:30 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).Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it aboutPreparation of standard solution: For 100/ 500/15 mg Tablets:30 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).Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50.0 mL volumetric flask, add to itPreparation of standard solution: For 100mg Tablets:about 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentrationof Aceclofenac is about 2000µg/mL) 473 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • 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).Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. WeighPreparation of test solution: For 100/ 500 / 500 mg Tablets:accurately 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.Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. WeighPreparation of test solution: For 100/ 325 / 250 mg Tablets:accurately 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.Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. WeighPreparation of test solution: For 100/ 500 / 15 mg Tablets:accurately 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.Equilibrate the column with mobile phase for sufficient time until stable baseline is obtained. Inject blank, standard andProcedure:sample 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. % content= Test solution area x Std. Dilution factor x Std. Potency Standard solution area x Test dilution factorCalculation: RESULTS AND DISCUSSIONStandard solution was prepared as per the proposed test method and injected into the HPLC system. The results of theSystem suitability:system suitability assessment for initial validation study parameters were tabulated in Table 1. 474 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 1: System suitability Sr. Observations System Suitability parameter Limits The % RSD of peak area response for No. Aceclofenac Paracetamol Chlorzoxazone 0.3 0.1 0.2 NMT 2.0 five replicate injections of standard 1 Theoretical plates 2963 1506 3986 NLT 1000 Tailing factor 2.0 1.8 1.9 NMT 2.0 2 3Precision Studies:Standard solution of working standard was prepared as per the proposed test procedure for repeatability studies. FiveSystem Precision:replicate 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 Precision Area Response 3481243 9580728 14147918 Injection No. Aceclofenac Paracetamol Chlorzoxazone 3480070 9596079 14192275 1 3474685 9605600 14200613 2 3456683 9587599 14148073 3 3462315 9597724 14154756 4 3470999 9593546 14168727 5 10967.78 9602.586 25622.336 Average 0.3 0.1 0.2 SD % RSDSix test preparations were prepared as per the proposed test method for individual test preparation. All individual testMethod Precision:preparations 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 Studies % Assay 98.4 95.8 103.4 Sr. No Aceclofenac Paracetamol Chlorzoxazone 98.1 95.9 103.4 1 98.7 95.8 103.5 2 98.6 96.1 103.9 3 98.2 95.9 103.5 4 98.8 95.6 103.2 5 98.5 95.9 103.5 6 0.2805 0.1643 0.2317 Average 0.3 0.2 0.2 SD % RSDSix test preparations were prepared as per the proposed test method for individual test preparation and injected into theRuggedness (Intermediate precision):different 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. 475 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 4: Ruggedness % Assay 98.8 95.8 103.8 Sr. No Aceclofenac Paracetamol Chlorzoxazone 98.4 95.7 103.7 1 98.6 95.8 103.8 2 98.8 96.1 104.0 3 98.9 96.0 103.9 4 98.9 96.0 103.9 5 98.7 95.9 103.9 6 0.1966 0.1549 0.1049 Average 0.2 0.2 0.1 SD % RSD Table 5: Ruggedness data evaluation % Assay 98.4 98.8 95.8 95.8 103.4 103.8 Sr. No Aceclofenac Paracetamol Chlorzoxazone 98.1 98.4 95.9 95.7 103.4 103.7 1 98.7 98.6 95.8 95.8 103.5 103.8 2 98.6 98.8 96.1 96.1 103.9 104.0 3 98.2 98.9 95.9 96.0 103.5 103.9 4 98.8 98.9 95.6 96.0 103.2 103.9 5 6 Overall 98.6 95.9 103.7 Average Overall SD 0.2697 0.1545 0.2570 Overall 0.3 0.2 0.2 % RSDLinearity and Range:The linearity studies of detector response for analytes were evaluated in the concentration range from about 50% of lowerLinearity of Detector Response:strength 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 Chlorzoxazone Sr. No Concentration Area Concentration Area Concentration Area 29.9976 1051778 150.4635 2644943 120.2281 3603632 (g/mL) response (g/mL) response (g/mL) response 59.9951 2105682 200.6180 3519271 200.3802 6047772 1 75.9938 2451169 401.2361 7010633 320.6083 9611741 2 99.9919 3503313 501.5451 8745948 500.9505 14946798 3 109.9911 3849570 551.6996 9595498 601.1406 17884943 4 119.9903 4187013 626.9314 10901381 721.3687 21323730 5 159.9870 5600698 752.3177 13044334 761.4448 22494050 6 0.999 1.000 1.000 7 Correlation 35288.4907 17291.5590 29427.4152 Coefficient 56878.5049 55950.5535 139465.4305 Slope Intercept 476 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • 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. TheLinearity of Test Method (Inferred from Accuracy):results 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. Table 7: Linearity of Test method (Inferred from accuracy)For 100/500/500 mg Tablets: Aceclofenac Paracetomol Chlorzoxazone Recovery level Amount Amount Amount Amount Amount Amount Recovered Added Recovered Added Recovered 79.4 81.0 397.2 401.0 397.1 403.0 Added (mg) (mg) (mg) (mg) (mg) (mg) 99.6 101.0 495.8 498.0 495.6 502.0 80% 119.6 121.0 594.8 590.0 595.0 594.0 100% 1.000 0.9999 0.9997 120% Correlation 0.995 0.9565 0.9651 Coefficient 1.9627 21.9933 21.0867 Slope InterceptPrecision at lower and higher extreme range concentration for Aceclofenac (29.9976 µg/ml and 159.9870 µg/ml),Precision at Lower and Higher extreme concentrations: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 linearity Area Responses Area Responses Aceclofenac Area Responses Paracetamol Chlorzoxazone Injection Number Lower level Higher level Lower level Higher level Lower level Higher level 1051198 5593151 2654481 13053921 3607531 22487458 (25%) (150%) (25%) (150%) (25%) (150%) 1053062 5599304 2655088 13029744 3605991 22475748 1 1052477 5593984 2646499 13073632 3602871 22554993 2 1051003 5598529 2637079 13028568 3600717 22472092 3 1052376 5602959 2635339 13056944 3598395 22506195 4 1050552 5616261 2641174 13023193 3606288 22467814 5 1051778 5600698 2644943 13044334 3603632 22494050 6 993.4749 8438.0317 8539.324 20088.1667 3586.7515 32895.1346 Average 0.1 0.2 0.3 0.2 0.1 0.1 SD % RSDAn accuracy study was conducted by spiking the known amount of analytes in the equivalent weight of placebo. AccuracyAccuracy:study 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. 477 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 9: Accuracy of Aceclofenac Aceclofenac Recovery Level Amount Amount Average 79.4 80.5 101.4 % Recovery % RSD Added (mg) Recovered (mg) Recovery (%) 79.4 80.9 101.9 101.5 0.4 79.5 80.4 101.1 80% 99.6 101.7 102.1 99.6 101.1 101.5 101.7 0.3 99.6 101.2 101.6 100% 119.6 119.8 100.2 119.6 121.3 101.4 101.3 1.0 119.6 122.4 102.3 120% Table 10: Accuracy Paracetamol Overall 101.5 0.6 Paracetamol Recovery Amount Amount Average 397.1 399.9 100.7 Level % Recovery % RSD Added (mg) Recovered (mg) Recovery (%) 397.1 402.6 101.4 101.0 0.4 397.3 400.3 100.8 80% 495.8 500.1 100.9 495.8 495.9 100.0 100.3 0.5 495.9 496.5 100.1 100% 594.8 582.6 97.9 594.8 590.9 99.3 99.2 1.3 594.8 597.3 100.4 120% Table 11: Accuracy of Chlorzoxazone Overall 100.2 1.0 Chlorzoxazone Recovery Amount Amount Average 397.1 403.6 101.6 Level % Recovery % RSD Added (mg) Recovered (mg) Recovery (%) 397.1 405.6 102.1 101.6 0.5 397.2 401.1 101.0 80% 495.6 502.6 101.4 495.7 502.0 101.3 101.4 0.1 495.6 502.4 101.4 100% 595.0 588.2 98.9 595.0 591.4 99.4 99.8 1.2 595.0 601.3 101.1 120% Overall 100.9 1.0Robustness:Mobile phase Flow rate as per proposed analytical method is 1.0 ml/min. Change in flow rate by –10% = 0.9 ml/min. Change inEffect of variation in Flow rate of mobile phase (±10%):flow 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. 478 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 12: System suitability of change in Flow Rate Sr. Observations The % RSD of Aceclofenac 0.1 0.1 0.2 System Suitability parameter Limits No. As Such - 10% + 10% peak area Paracetamol 0.1 0.1 0.2 response for five NMT 2.0 replicate Chlorzoxazone 0.1 0.2 0.3 1 injections Aceclofenac 6209 6301 6158 Theoretical plates Paracetamol 2559 2697 2413 NLT 1000 Chlorzoxazone 10476 10660 9988 2 Aceclofenac 2.0 2.0 2.0 Tailing factor Paracetamol 1.4 1.4 1.3 NMT 2.0 Chlorzoxazone 1.1 1.1 1.1 3 The Column Oven temperature as per proposed analytical method is 30°C. Change in Column oven Temperature by –5°C = Effect of variation in Column oven temperature (±5°C): 25°C. Change in Column oven Temperature by +5°C = 35°C. The effect due to change in Column oven temperature on the system suitability parameters are compared. Results are tabulated in Table 13. Table 13: System suitability of change in Column Oven temperature Sr. Observations System Suitability parameter Limits Aceclofenac 0.1 0.1 0.5 No. The % RSD of peak As Such -5°C + 5°C area response for five Paracetamol 0.1 0.0 0.1 NMT 2.0 replicate injections Chlorzoxazone 0.1 0.0 0.1 1 Aceclofenac 6209 5881 6531 Theoretical plates Paracetamol 2559 2434 2497 NLT 1000 Chlorzoxazone 10476 9784 9947 2 Aceclofenac 2.0 2.0 2.0 Tailing factor Paracetamol 1.4 1.4 1.4 NMT 2.0 Chlorzoxazone 1.1 1.1 1.1 3The Organic phase ratio of mobile phase as per proposed analytical method is 0.2% v/v Orthophosphoric acid: Acetonitrile isEffect of variation in organic phase composition (± 10%):67:33 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 composition Sr. Observations The % RSD of peak Aceclofenac 0.1 0.2 0.1 System Suitability parameter Limits No. As Such -5% + 5% area response for five Paracetamol 0.2 0.1 0.2 NMT 2.0 replicate injections Chlorzoxazone 0.4 0.1 0.1 1 Aceclofenac 5973 6363 5176 Theoretical plates Paracetamol 2526 2216 2581 NLT 1000 Chlorzoxazone 8420 8537 7227 2 Aceclofenac 1.6 1.3 1.4 Tailing factor Paracetamol 1.4 1.3 1.4 NMT 2.0 Chlorzoxazone 1.1 1.1 1.1 3 479 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490The pH of mobile phase A as per proposed analytical method is 6.0. Change in pH of mobile phase A by – 0.2 units is 5.8Effect of variation in Mobile phase pH (± 0.2 Units):Change in pH of mobile phase A by + 0.2 units is 6.2 The effect due to change in pH of mobile phase A on the systemsuitability parameters are compared. Results are tabulated in Table 15. Table 15: System suitability of change in mobile phase pH Sr. Observations System Suitability parameter Limits The % RSD of Aceclofenac 0.2 0.1 0.0 No. As Such - 0.2 units + 0.2units peak area Paracetamol 0.1 0.1 0.0 response for five NMT 2.0 replicate Chlorzoxazone 0.2 0.1 0.1 1 injections Aceclofenac 6470 6225 6333 Theoretical plates Paracetamol 2055 2100 2107 NLT 1000 Chlorzoxazone 9806 9473 9646 2 Aceclofenac 1.9 1.9 1.9 Tailing factor Paracetamol 1.3 1.4 1.4 NMT 2.0 Chlorzoxazone 1.1 1.1 1.1 3 Standard solutions and sample solution were prepared. Some portion of above solutions was filtered through 0.45 Filter Interference studies: membrane filter. Both the samples further diluted as per proposed method and injected in to the HPLC system. For comparison the % area difference was calculated between unfiltered and filtered solutions. The data tabulated in Table 16, Table 16: Filter paper details 17, 18 and 19. Nylon MDI Syringe 0.45µ 25 mm SN0850 Type Make Micron size Diameter Lot No Table 17: Filter paper interference study Aceclofenac Aceclofenac Average Standard Area Average Test Area Prep. No. % % STD-1 % Diff STD-2 Test-1 % Diff Test-2 3551905 NA 3524335 NA 3595532 NA 3591554 NA Diff Diff 3548229 0.1 3563626 1.1 3588334 0.2 3581025 0.3 Unfiltered 0.45µFilter Table 18: Filter paper interference study Paracetamol Paracetamol Average Standard Area Average Test Area Prep. No. % % 9556575 NA 9525387 NA 9730520 NA 9753048 NA STD-1 STD-2 % Diff Test-1 % Diff Test-2 Diff Diff 9512533 0.5 9564122 0.4 9720541 0.1 9713146 0.4 Unfiltered 0.45µFilter 480 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 19: Filter paper interference study Chlorzoxazone Chlorzoxazone Average Standard Area Average Test Area Prep. No. % % STD-1 % Diff STD-2 Test-1 % Diff Test-2 14171478 NA 14113560 NA 15017843 NA 15040877 NA Diff Diff 14143527 0.2 14114024 0.0 14979503 0.3 14994880 0.3 Unfiltered 0.45µFilterTest preparation was prepared as per the test method and injected into HPLC system. The peak purity result wasPeak Purity results of Sample As such:evaluated 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 tablets Peak purity results Parameter Aceclofenac Chlorzoxazone Paracetamol Purity-1 angle 0.071 0.271 2.092 Purity-1 threshold 0.251 0.282 6.619 Purity Flag No No No Table 21: Sample As such Lower Strength 100/500/15 mg tablets Peak purity results Parameter Aceclofenac Paracetamol Purity-1 angle 0.057 2.171 Purity-1 threshold 0.232 6.775 Purity Flag No NoThe stress degradation study was carried out on the sample preparations (100/500/500 mg and 100/500/15 mg strength)Forced Degradation studies: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. Table 22: % degradation data for 100/500/500 mg tablets Aceclofenac Paracetamol Chlorzoxazone Stress Condition % % % Acid degradation degradation degradation degradation 16.2 0.1 3.2 0.1N HCl, 1hr at 60°C Alkali degradation 95.1 9.9 7.0 1N NaOH, 1hr at 80°C Peroxide degradation 17.5 0.5 1.9 6% H2O2, 2hrs at 80°C Thermal degradation 11.3 1.4 1.4 2 hrs at 80°C Photolytic degradation 12.0 1.4 1.6 1.2 m.lux hrs 481 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 23: % degradation data for 100/500/15 mg tablets Aceclofenac Paracetamol Stress Condition % % Acid degradation 0.1N HCl, 1hr at 60°C 10.7 3.6 degradation degradation Alkali degradation 1N NaOH, 1hr at 80°C 98.6 19.2 Peroxide degradation 6% H2O2, 2hrs at 80°C 17.4 2.5 Thermal degradation 2 hrs at 80°C 8.2 3.0 Photolytic degradation 1.2 m.lux hrs 8.2 3.0Solution Stability Studies:The mobile phase was prepared as per the proposed test method and kept at room temperature for a period of two days inMobile phase stability at room temperature:well 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 temperature Observations The % RSD of peak area Aceclofenac 0.2 0.1 Sr. No. System Suitability parameter Limits Initial Day-1 response for five replicate Paracetamol 0.2 0.2 NMT 2.0 injections Chlorzoxazone 0.1 0.1 1 Aceclofenac 6737 6704 Theoretical plates Paracetamol 3013 3227 NLT 1000 Chlorzoxazone 10870 10725 2 Aceclofenac 1.9 1.8 Tailing factor Paracetamol 1.3 1.2 NMT 2.0 Chlorzoxazone 1.0 1.0 3Standard and test preparations were prepared as per the proposed test method and the stock solutions were kept at roomAnalytical Solution Stability (Standard and Test preparation) at room temperature:temperature. 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, 26 Table 25: Stability of Aceclofenac Standard and test solution at room temperatureand 27. Aceclofenac Standard Aceclofenac Test 3549534 NA 3598615 NA Time (Hours) Area Response % Difference Area Response % Difference 3539237 0.3 3578216 0.6 Initial 3540881 0.2 3609559 0.3 4 Hrs 35 min 3559542 0.3 3597509 0.0 8 Hrs 3546836 0.1 3575222 0.7 12 Hrs 15 min 3540089 0.3 3575272 0.6 16 Hrs 20 min 3557104 0.2 3571046 0.8 20 Hrs 25 min 3528644 0.6 3562606 1.0 23 Hrs 3532159 0.5 3581784 0.5 24 Hrs 30 min 3581358 0.9 3581157 0.5 29 Hrs 3630421 2.3 3581119 0.5 33 Hrs 40 Hrs 50 min 482 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 26: Stability of Paracetamol Standard and test solution at room temperature Paracetamol Standard Paracetamol Test 9533772 NA 9674499 NA Time (Hours) Area Response % Difference Area Response % Difference 9485553 0.5 9681641 0.1 Initial 9529119 0.0 9720005 0.5 4 Hrs 35 min 9562369 0.3 9713140 0.4 8 Hrs 9558274 0.3 9703185 0.3 12 Hrs 15 min 9551274 0.2 9705705 0.3 16 Hrs 20 min 9554327 0.2 9673281 0.0 20 Hrs 25 min 9551111 0.2 9659821 0.2 23 Hrs 9541020 0.1 9661378 0.1 24 Hrs 30 min 9619965 0.9 9674547 0.0 29 Hrs 9760962 2.4 9661793 0.1 33 Hrs Table 27: Stability of Chlorzoxazone Standard and test solution at room temperature 40 Hrs 50 min Chlorzoxazone Standard Chlorzoxazone Test 14101688 NA 14905697 NA Time (Hours) Area Response % Difference Area Response % Difference 14074515 0.2 14896823 0.1 Initial 14105493 0.0 15018775 0.84 Hrs 35 min 14182706 0.6 14980781 0.58 Hrs 14175597 0.5 14922091 0.112 Hrs 15 min 14185354 0.6 14925356 0.116 Hrs 20 min 14165758 0.5 14885819 0.120 Hrs 25 min 14150058 0.3 14871542 0.223 Hrs 14064098 0.3 14908388 0.024 Hrs 30 min 14290784 1.3 14911371 0.029 Hrs 14499323 2.8 14930540 0.233 Hrs40 Hrs 50 min BLANK CHROMATOGRAM 483 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 STANDARD CHROMATOGRAM TEST CHROMATOGRAM LINEARITY OF DETECTOR RESPONSE OF ACECLOFENAC 484International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 LINEARITY OF DETECTOR RESPONSE OF PARACETAMOL LINEARITY OF DETECTOR RESPONSE OF CHLORZOXAZONE LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF ACECLOFENAC 485International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF PARACETAMOL LINEARITY GRAPH (INFERRED FROM ACCURACY STUDIES) OF CHLORZOXAZONE AS SUCH SAMPLE CHROMATOGRAM HIGHER STRENGTH 486International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 AS SUCH SAMPLE PURITY PLOT HIGHER STRENGTH AS SUCH SAMPLE CHROMATOGRAM LOWER STRENGTH 487International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 AS SUCH SAMPLE PURITY PLOT LOWER STRENGTH ACID DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH 488International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 ALKALI DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH PEROXIDE DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH THERMAL DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH 489International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012
    • Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 PHOTOLYTIC DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH The complete study results reveals that the developed and validated RP-HPLC method is accurate, precise, robust and CONCLUSIONstability indicating. This method has wide applicability and useful for regular quality control analysis of formulation samples.1. Granberg RA, Rasmuson AC, Solubility of paracetamol in pure solvents, Journal of Chemical & Engineering Data, 1999, 44 REFERENCES (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 likely mechanisms 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 and Availability, 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. 490 International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012