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Hari Kishan Reddy Ganthi et al.

, IJSID, 2012, 2 (4), 471-490

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries


Research Article

An International peer Review Journal for Science

Available online through www.ijsidonline.info

SINGLE RP-HPLC METHOD FOR THE QUANTIFICATION OF ACECLOFENAC, PARACETAMOL AND CHLOROZOXAZONE IN FORMULATIONS Hari kishan Reddy Ganthi1*, Hanimi Reddy Bapatu2, Maram Ravi Kumar3, Useni Reddy Mallu1, Dr. Reddys Laboratories Ltd, Bachupally, Hyd-72, India.
ABSTRACT Objectives: To develop a single RP-HPLC method for determination of Aceclofenac, Paracetamol and Chlorozoxazone contents in formulation. was at 275 nm. Methods: Chromatographic separation was achieved on Inertsil ODS 3V, 150 x4.6mm, 5 column. Mobile phase composed of phosphate b u f f e r Results: High resolution was achieved with the simple mobile phase composition and 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 retention time of Paracetamol, Chlorozoxazone, and Aceclofenac are about 2.1min, 8.8min Address: Name: Hari Kishan Reddy Ganthi Place: Sri Krishnadevaraya University Anantapur, AP, India E-mail: kishangan05ster@gmail.com concentration in the range 150.4 to 752.3 ppm for Paracetamol, 120.2 to 761.4 ppm for value of all activeINTRODUCTION limit (0.999). ingredients within the reproducibility and ruggedness and it is applicable for regular analysis. and 20.7min, respectively. The area of all ingredient peaks were a linear function of Chlorozoxazone and 29.9 to 159.9 ppm for Aceclofenac and the correlation co-efficient Conclusion: Proposed HPLC method was validated with specificity, linearity, accuracy, Keywords: Aceclofenac, Paracetamol, Chlorozoxazone and RP-HPLC method.
1Dept.

JNT University, Kukatpally, Hyderabad, AP, India-500072.; 3 AR&D, Custom Pharmaceutical Services,

of Chemistry, Sri Krishnadevaraya University, Anantapur, AP, India; 2Department of Chemistry,

Received: 14-08-2012 Accepted: 17-10-2012


*Corresponding Author

INTRODUCTION

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Paracetamol (acetaminophen) is one of the most popular over-the-counter (OTC) analgesic and antipyretic drugs. INTRODUCTION Paracetamol (1-8) is available in different dosage forms: tablet, capsules, drops, elixirs, suspensions and suppositories. people with porphyria or breast-feeding mothers, and is not recommended for children. Aceclofenac is a non-steroidal anti-inflammatory drug (NSAID). It is used for the relief of pain and inflammation in Chlorzoxazone is used to relieve pain and stiffness caused by muscle strains and sprains. It is used in combination with physical 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. dosage forms. Paracetamol Chlorozoxazone Aceclofenac

rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. The dose is 100 mg twice daily. It should not be given to

Chemical structures of all ingredients were represented in figure-1.All three ingredients are available in liquid pharmaceutical

study is to develop a single RP-HPLC method for the estimation of Paracetamol, Aceclofenac and Chlorozoxazone in formulations and the developed and validated method is simple, novel, rugged and linear. MATERIALS AND METHODS Selection of mobile phase: ratio. Various buffer salts, pH values were tried with different organic solvents (acetonitrile or methanol) for the optimization of Ortho phosphoric acid, triethyl amine (AR Grade) was procured from S.D fine chemicals. High pure (NLT 98.5%) standards Chemicals and reagents: Spectrochem Pvt. Ltd. Water is prepared by mili Q system (Milli-pore). Filtered through 0.45m membrane filter and degassed. Diluent: Mobile phase. Column HPLC conditions: Wavelength Injection volume: : :

All ingredients have reported methods for individual and other combination products, but the objective of the present

Figure-1: Chemical structure of all ingredients

mobile phase. Finally well shaped and high resolution was achieved with pH 6.0 phosphate buffer and acetonitrile at 67:33 v/v (Aceclofenac, Paracetamol and Chlorozoxazone) were used for this study. HPLC grade acetonitrile were procured from Buffer preparation: Diluted 2.0mL of ortho phosphoric acid to 1000 mL of water. Adjusted to pH 6.0 with triethyl amine Mobile Phase: Mixed the buffer and acetonitrile in the ration 67:33 v/v. Inertsil ODS 3V (150X4.6mm, 5m) 275nm 20 L

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Flow rate Run time

Temperature

Mobile phase

: : : :

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 1.0 mL/min 30C 30min

Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about Preparation of standard solution: For 100/ 500 / 500 mg Tablets: Aceclofenac is about 2000g/mL) 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration of in to 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up Paracetamol is about 500g/mL and concentration of Chlorzoxazone is about 500g/mL ). Preparation of standard solution: For 100/ 325/ 250 mg Tablets: Aceclofenac is about 2000g/mL) the mark with mobile phase Accurately weigh and transfer 100 mg of Paracetamol working standard, and 100 mg of Chlorzoxazone working standard

Buffer and Acetonitrile 67:33 v/v

to the mark with mobile phase and mix well. (Concentration of Aceclofenac is about 100 g/mL, concentration of 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration of to 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to Paracetamol is about 325g/mL and concentration of Chlorzoxazone is about 250g/mL). Preparation of standard solution: For 100/ 500 mg Tablets: Aceclofenac is about 2000g/mL)

Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about Accurately weigh and transfer 65 mg of Paracetamol working standard, and 50 mg of Chlorzoxazone working standard in

Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration of Accurately weigh and transfer 100 mg of Paracetamol working standard, in to 200 mL volumetric flask, add to it about 10 (Concentration of Aceclofenac is about 100 g/mL, concentration of Paracetamol is about 500g/mL). Preparation of standard solution: For 100/ 500/15 mg Tablets: Aceclofenac is about 2000g/mL) mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to the mark with mobile phase and mix well. 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration of (Concentration of Aceclofenac is about 100 g/mL, concentration of Paracetamol is about 500g/mL). Preparation of standard solution: For 100mg Tablets: of Aceclofenac is about 2000g/mL)

and mix well. (Concentration of Aceclofenac is about 100 g/mL, concentration of

Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50 mL volumetric flask, add to it about Accurately weigh and transfer 100 mg of Paracetamol working standard, in to 200 mL volumetric flask, add to it about 10 mL of above Aceclofenac stock solution and sonicate to dissolve, dilute up to the mark with mobile phase and mix well. Accurately weigh and transfer about 100 mg of Aceclofenac working standard into 50.0 mL volumetric flask, add to it International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012

about 30 mL of acetonitrile and sonicate to dissolve, dilute up to the mark with acetonitrile and mix well. (Concentration

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Dilute the 10 mL of above Aceclofenac stock solution to 200 mL with mobile phase and mix well. (Concentration of Aceclofenac is about 100 g/mL). Preparation of test solution: For 100/ 500 / 500 mg Tablets: accurately the powder equivalent to 500 mg of Paracetamol into diluent.

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490

Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weigh acetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minutes

with intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix. Preparation of test solution: For 100/ 325 / 250 mg Tablets: accurately the powder equivalent to 325 mg of Paracetamol into diluent.

Filter the solution through 0.45 nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL with

a 200 mL volumetric flask, add to it 20 ml of

Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weigh with intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix. Filter the solution through 0.45 nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL with Preparation of test solution: For 100/ 500 / 15 mg Tablets: accurately the powder equivalent to 500 mg of Paracetamol into diluent. Accurately weigh not less than 20 tablets and determine the average weight. Crush the tablets to fine powder. Weigh acetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minutes Prepare the test solutions in duplicate. Procedure: Filter the solution through 0.45 nylon membrane filter. Further dilute 5 mL of the supernatant solution to 25 mL with a 200 mL volumetric flask, add to it 20 ml of

acetonitrile and sonicate to disperse the content. Add to it 130 ml of mobile phase and sonicate to dissolve for 15 minutes

with intermittent shaking. Allow the solution cool to room temperature. Dilute to the volume with mobile phase and mix.

a 200 mL volumetric flask, add to it 20 ml of

Equilibrate the column with mobile phase for sufficient time until stable baseline is obtained. Inject blank, standard and sample preparation filter through 0.45 nylon filter. Inject blank (diluent) in single, standard preparation in five replicates, and each test preparation into the chromatographic system and record the chromatograms. Inject standard preparation as a bracketing after every six injections of test preparations. Evaluate the system suitability parameters from the standard chromatograms. The order of elution is Paracetamol, Chlorzoxazone and Aceclofenac. Calculation: % content= Test solution area x Std. Dilution factor x Std. Potency Standard solution area x Test dilution factor RESULTS AND DISCUSSION

Standard solution was prepared as per the proposed test method and injected into the HPLC system. The results of the System suitability: system 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 2012

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 1: System suitability Sr. No. 1 2 The % RSD of peak area response for five replicate injections of standard Theoretical plates System Suitability parameter Aceclofenac 2963 2.0 0.3 Observations Paracetamol 1506 1.8 0.1 Chlorzoxazone 3986 1.9 0.2 NMT 2.0 NMT 2.0 Limits

3 Tailing factor Precision Studies: System Precision:

NLT 1000

Standard solution of working standard was prepared as per the proposed test procedure for repeatability studies. Five 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. Injection No. 1 2 3 4 5 Average SD % RSD Method Precision: Table 2: System Precision Aceclofenac 3481243 3480070 3474685 3456683 3462315 3470999 10967.78 0.3 Area Response Paracetamol 9580728 9596079 9605600 9587599 9597724 9593546 9602.586 0.1 Chlorzoxazone 14147918 14192275 14200613 14148073 14154756 14168727 25622.336 0.2

Six test preparations were prepared as per the proposed test method for individual test preparation. All individual test preparations were injected into the HPLC system as per the test method. The %assay results were calculated for each individual test sample, along with average assay and % RSD for the six preparations. The results are tabulated in Table 3. Table 3: Method Precision Studies Aceclofenac 98.4 98.1 98.7 98.6 98.2 98.8 98.5 0.2805 0.3 Sr. No 1 2 3 4 5 6 Average SD % RSD Ruggedness (Intermediate precision): % Assay Paracetamol 95.8 95.9 95.8 96.1 95.9 95.6 95.9 0.1643 0.2 Chlorzoxazone 103.4 103.4 103.5 103.9 103.5 103.2 103.5 0.2317 0.2

Six test preparations were prepared as per the proposed test method for individual test preparation and injected into the different HPLC system by the different analyst using different make of HPLC Column at different day. the six preparations were calculated. The results are tabulated in Table 4.

The %assay results were calculated for each of the sample for each of the variability. Average assay values and % RSD for International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 4: Ruggedness Sr. No 1 2 3 4 5 6 Average SD % RSD Sr. No 1 2 3 4 5 6 Overall Average Overall SD Overall % RSD Linearity and Range: % Assay Aceclofenac Paracetamol 98.8 95.8 98.4 95.7 98.6 95.8 98.8 96.1 98.9 96.0 98.9 96.0 98.7 95.9 0.1966 0.1549 0.2 0.2 Table 5: Ruggedness data evaluation % Assay Paracetamol 95.8 95.8 95.9 95.7 95.8 95.8 96.1 96.1 95.9 96.0 95.6 96.0 95.9 0.2 0.1545 Chlorzoxazone 103.8 103.7 103.8 104.0 103.9 103.9 103.9 0.1049 0.1

Aceclofenac 98.4 98.8 98.1 98.4 98.7 98.6 98.6 98.8 98.2 98.9 98.8 98.9 98.6 0.3 0.2697

Chlorzoxazone 103.4 103.8 103.4 103.7 103.5 103.8 103.9 104.0 103.5 103.9 103.2 103.9 103.7 0.2 0.2570

The linearity studies of detector response for analytes were evaluated in the concentration range from about 50% of lower Linearity of Detector Response: strength to 150% of the higher strength of the targeted concentration. The diluted standard solutions were prepared from stock solution in the above range and analysed using proposed analytical method by injecting each level in to the system. calculated. The results are tabulated in Table 6. Sr. No 1 2 3 4 5 6 7 Correlation Coefficient Slope Intercept Table6: Linearity of Detector Response: Paracetamol Concentration Area response (g/mL) 150.4635 2644943 200.6180 3519271 401.2361 7010633 501.5451 8745948 551.6996 9595498 626.9314 10901381 752.3177 13044334 17291.5590 55950.5535 1.000

The Linearity graph of average area response verses concentration was plotted and the correlation coefficient was Aceclofenac Concentration Area response (g/mL) 29.9976 1051778 59.9951 2105682 75.9938 2451169 99.9919 3503313 109.9911 3849570 119.9903 4187013 159.9870 5600698 35288.4907 56878.5049 0.999 Chlorzoxazone Concentration Area response (g/mL) 120.2281 3603632 200.3802 6047772 320.6083 9611741 500.9505 14946798 601.1406 17884943 721.3687 21323730 761.4448 22494050 29427.4152 139465.4305 1.000

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 The results obtained in accuracy study were further interpreted for the evaluation of linearity of the test method. The Linearity of Test Method (Inferred from Accuracy): correlation coefficient for set of data was evaluated. The results are tabulated in Table 7. For 100/500/500 mg Tablets: Table 7: Linearity of Test method (Inferred from accuracy) Aceclofenac Recovery level 80% 100% 120% Correlation Coefficient Slope Amount Added (mg) 119.6 79.4 99.6 Amount Recovered (mg) 81.0 101.0 121.0 Paracetomol Amount Added (mg) 397.2 495.8 594.8 Amount Recovered (mg) 401.0 498.0 590.0 results of average of mg added at each spiked level was plotted against average mg recovered at each accuracy level and the

Chlorzoxazone Amount Added (mg) 397.1 495.6 595.0 Amount Recovered (mg) 403.0 502.0 594.0

Intercept 1.9627 Precision at Lower and Higher extreme concentrations:

1.000 0.995

Precision at lower and higher extreme range concentration for Aceclofenac (29.9976 g/ml and 159.9870 g/ml), Paracetamol (150.4635 g/ml and 752.3177 g/ml), and Chlorzoxazone (120.2281 g/ml and 761.4448 g/ml) of levels. % RSD for peak responses for both the level was evaluated. The results are tabulated in Table 8. Injection Number 1 2 3 4 5 6 Average SD % RSD Accuracy: Area Responses Aceclofenac Lower level (25%) 1051198 1053062 1052477 1051003 1052376 1050552 1051778 993.4749 0.1 Table 8: Precision at Lower and Higher extreme Levels of linearity Area Responses Paracetamol Lower level (25%) 2654481 2655088 2646499 2637079 2635339 2641174 2644943 8539.324 0.3 Higher level (150%) 13053921 13029744 13073632 13028568 13056944 13023193 13044334 20088.1667 0.2 Higher level (150%) 5593151 5599304 5593984 5598529 5602959 5616261 5600698 8438.0317 0.2

21.9933

0.9999 0.9565

21.0867

0.9997 0.9651

Linearity levels were determined as per proposed method by injecting six replicate injections of standards for both the Area Responses Chlorzoxazone Lower level Higher level (25%) (150%) 3607531 22487458 3605991 22475748 3602871 22554993 3600717 22472092 3598395 22506195 3606288 22467814 3603632 22494050 3586.7515 32895.1346 0.1 0.1

An accuracy study was conducted by spiking the known amount of analytes in the equivalent weight of placebo. Accuracy study was conducted in triplicate at three different levels, (80%, 100% and 120% of target concentration). The samples each spike level was also established. The results are tabulated in Table 9, 10 and 11. were analyzed as per the proposed test procedure and the % recovery for each spike level was calculated. The precision at

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 9: Accuracy of Aceclofenac 101.4 101.9 101.1 102.1 101.5 101.6 100.2 101.4 102.3 Overall Table 10: Accuracy Paracetamol % Recovery % Recovery

Recovery Level 80% 100% 120%

Aceclofenac Amount Added (mg) 79.4 79.4 79.5 99.6 99.6 99.6 119.6 119.6 119.6 Paracetamol Amount Added (mg) 397.1 397.1 397.3 495.8 495.8 495.9 594.8 594.8 594.8 Chlorzoxazone Amount Added (mg) 397.1 397.1 397.2 495.6 495.7 495.6 595.0 595.0 595.0

Amount Recovered (mg) 80.5 80.9 80.4 101.7 101.1 101.2 119.8 121.3 122.4

Average Recovery (%) 101.5 101.7 101.3

% RSD 0.4 0.3 1.0

101.5

0.6

Recovery Level 80% 100% 120%

Amount Recovered (mg) 399.9 402.6 400.3 500.1 495.9 496.5 582.6 590.9 597.3

Recovery Level 80% 100% 120% Robustness:

100.7 101.4 100.8 100.9 100.0 100.1 97.9 99.3 100.4 Overall Table 11: Accuracy of Chlorzoxazone % Recovery 101.6 102.1 101.0 101.4 101.3 101.4 98.9 99.4 101.1 Overall

Average Recovery (%) 101.0 100.3 99.2

% RSD 0.4 0.5 1.3

100.2

1.0

Amount Recovered (mg) 403.6 405.6 401.1 502.6 502.0 502.4 588.2 591.4 601.3

Average Recovery (%) 101.6 101.4 99.8

% RSD 0.5 0.1 1.2

100.9

1.0

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 in Effect of variation in Flow rate of mobile phase (10%): Results are tabulated in Table 12. flow rate by +10% = 1.1 ml/min. The effect due to change in flow rate on the system suitability parameters are compared.

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Table 12: System suitability of change in Flow Rate Observations System Suitability parameter As Such - 10% The % RSD of Aceclofenac 0.1 0.1 peak area Paracetamol 0.1 0.1 1 response for five replicate Chlorzoxazone 0.1 0.2 injections Aceclofenac 6209 6301 2 Theoretical plates Paracetamol 2559 2697 Chlorzoxazone 10476 10660 Aceclofenac 2.0 2.0 3 Tailing factor Paracetamol 1.4 1.4 Chlorzoxazone 1.1 1.1 Effect of variation in Column oven temperature (5C): Sr. No. system suitability parameters are compared. Results are tabulated in Table 13. Aceclofenac Paracetamol Chlorzoxazone Aceclofenac 2 Theoretical plates Paracetamol Chlorzoxazone Aceclofenac 3 Tailing factor Paracetamol Chlorzoxazone Effect of variation in organic phase composition ( 10%): 1 The % RSD of peak area response for five replicate injections Sr. No. System Suitability parameter 0.1 0.1 0.1 6209 2559 10476 2.0 1.4 1.1

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490

+ 10% 0.2 0.2 6158 2413 9988 2.0 1.3 1.1 0.3

Limits NMT 2.0

NLT 1000 NMT 2.0

The Column Oven temperature as per proposed analytical method is 30C. Change in Column oven Temperature by 5C = 25C. Change in Column oven Temperature by +5C = 35C. The effect due to change in Column oven temperature on the Table 13: System suitability of change in Column Oven temperature Observations 0.1 0.0 0.0 5881 2434 9784 2.0 1.4 1.1 -5C As Such

+ 5C

The Organic phase ratio of mobile phase as per proposed analytical method is 0.2% v/v Orthophosphoric acid: Acetonitrile is compared. Results are tabulated in Table 14. Sr. No. 1 2 3 The % RSD of peak area response for five replicate injections Theoretical plates Tailing factor Table 14: System suitability of change in organic phase composition Aceclofenac Paracetamol Chlorzoxazone Aceclofenac Paracetamol Chlorzoxazone Aceclofenac Paracetamol Chlorzoxazone As Such 0.1 0.2 0.4 5973 2526 8420 1.6 1.4 1.1 Observations -5% 0.2 0.1 0.1 6363 2216 8537 1.3 1.3 1.1

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 are

0.5 0.1 0.1 6531 2497 9947 2.0 1.4 1.1

Limits NMT 2.0 NMT 2.0

NLT 1000

System Suitability parameter

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+ 5% 0.1 0.2 0.1 5176 2581 7227 1.4 1.4 1.1

NLT 1000 NMT 2.0

NMT 2.0

Limits

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 The 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 5.8 Effect 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 system suitability parameters are compared. Results are tabulated in Table 15. Sr. No. System Suitability parameter Aceclofenac Paracetamol Table 15: System suitability of change in mobile phase pH Observations - 0.2 units 0.1 0.1 0.1 As Such 0.1 0.2 0.2

2 3

The % RSD of peak area response for five replicate injections Theoretical plates Tailing factor

+ 0.2units 0.0 0.1

Chlorzoxazone

0.0

Limits NMT 2.0

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 17, 18 and 19. Table 16: Filter paper details Micron size 0.45 comparison the % area difference was calculated between unfiltered and filtered solutions. The data tabulated in Table 16, Type Nylon Make MDI Syringe Diameter 25 mm Lot No SN0850

Aceclofenac Paracetamol Chlorzoxazone Aceclofenac Paracetamol Chlorzoxazone

6470 2055 9806 1.9 1.3 1.1

6225 2100 9473 1.9 1.4 1.1

6333 2107 9646 1.9 1.4 1.1

NLT 1000 NMT 2.0

Table 17: Filter paper interference study Aceclofenac

Prep. No. Unfiltered 0.45Filter

Aceclofenac Average Standard Area % STD-1 % Diff STD-2 Diff 3551905 NA 3524335 NA 3548229 0.1 3563626 1.1

Average Test Area 3595532 3588334 Test-1 % Diff NA 0.2 3591554 3581025 Test-2 % Diff NA 0.3

Prep. No. Unfiltered 0.45Filter

Paracetamol Average Standard Area % STD-1 STD-2 % Diff Diff 9556575 NA 9525387 NA 9512533 0.5 9564122 0.4

Table 18: Filter paper interference study Paracetamol 9730520 9720541 Test-1

Average Test Area % Diff NA 0.1 9753048 9713146 Test-2

% Diff NA 0.4

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 19: Filter paper interference study Chlorzoxazone 15017843 14979503 Test-1

Prep. No. Unfiltered 0.45Filter

Test preparation was prepared as per the test method and injected into HPLC system. The peak purity Peak Purity results of Sample As such: evaluated by Photo Diode Array Detector. The results are tabulated in Table 20 and 21. Parameter Purity-1 angle Purity-1 threshold Purity Flag Table 20: Sample As such Higher Strength 100/500/500 mg tablets Peak purity results Aceclofenac Chlorzoxazone 0.071 0.271 0.251 0.282 No No Paracetamol 2.092 6.619 No

Chlorzoxazone Average Standard Area % STD-1 % Diff STD-2 Diff 14171478 NA 14113560 NA 14143527 0.2 14114024 0.0

Average Test Area % Diff NA 0.3 15040877 14994880 Test-2 % Diff NA 0.3

result

was

Table 21: Sample As such Lower Strength 100/500/15 mg tablets Peak purity results Parameter Aceclofenac Purity-1 angle 0.057 Purity-1 threshold 0.232 Purity Flag No Paracetamol 2.171 6.775 No

The stress degradation study was carried out on the sample preparations (100/500/500 mg and 100/500/15 mg strength) Forced Degradation studies: characteristics of the method. The stress conditions and results were compiled in Table 22 and 23. Stress Condition Table 22: % degradation data for 100/500/500 mg tablets Aceclofenac % degradation 16.2 95.1 17.5 11.3 12.0 Paracetamol % degradation 0.1 9.9 0.5 1.4 1.4

of Tablet, and the degradation was evaluated by calculating the % degradation of in comparison with unstressed sample preparation. The degradation of 10-30% was tried by following stress conditions to prove the stability indicating Chlorzoxazone % degradation 3.2 7.0 1.9 1.4 1.6

Acid degradation 0.1N HCl, 1hr at 60C

Alkali degradation 1N NaOH, 1hr at 80C Peroxide degradation 6% H2O2, 2hrs at 80C Thermal degradation 2 hrs at 80C Photolytic degradation 1.2 m.lux hrs

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 Table 23: % degradation data for 100/500/15 mg tablets Aceclofenac % degradation 10.7 98.6 17.4 8.2 8.2

Acid degradation 0.1N HCl, 1hr at 60C Alkali degradation 1N NaOH, 1hr at 80C Peroxide degradation 6% H2O2, 2hrs at 80C Thermal degradation 2 hrs at 80C Photolytic degradation 1.2 m.lux hrs Solution Stability Studies:

Stress Condition

The mobile phase was prepared as per the proposed test method and kept at room temperature for a period of two days in Mobile phase stability at room temperature: well closed condition. The system suitability solutions were prepared and injected into the HPLC system at initially and changes like haziness or precipitation. System suitability results are tabulated in Table 24. Sr. No. System Suitability parameter Table 24: Mobile phase stability at room temperature

Paracetamol % degradation 3.6 19.2 2.5 3.0 3.0

periodically after 1day. The system suitability parameters were evaluated. The mobile phase was also observed for Physical Observations Initial Day-1 0.2 0.1 The % RSD of peak area Aceclofenac 1 response for five replicate Paracetamol 0.2 0.2 injections Chlorzoxazone 0.1 0.1 Aceclofenac 6737 6704 2 Theoretical plates Paracetamol 3013 3227 Chlorzoxazone 10870 10725 Aceclofenac 1.9 1.8 3 Tailing factor Paracetamol 1.3 1.2 Chlorzoxazone 1.0 1.0 Analytical Solution Stability (Standard and Test preparation) at room temperature: Limits

NLT 1000 NMT 2.0

NMT 2.0

Standard and test preparations were prepared as per the proposed test method and the stock solutions were kept at room temperature. The solutions were diluted freshly at each time and injected into the HPLC system at initially and at different time intervals. The % difference in area was calculated against the fresh standard solution injected at each time interval. and 27. Table 25: Stability of Aceclofenac Standard and test solution at room temperature Aceclofenac Standard Area Response 3549534 3539237 3540881 3559542 3546836 3540089 3557104 3528644 3532159 3581358 3630421 % Difference NA 0.3 0.2 0.3 0.1 0.3 0.2 0.6 0.5 0.9 2.3

The % difference in area response was evaluated against the initial assay value. The results are tabulated in Table 25, 26 Aceclofenac Test Area Response % Difference 3598615 NA 3578216 0.6 3609559 0.3 3597509 0.0 3575222 0.7 3575272 0.6 3571046 0.8 3562606 1.0 3581784 0.5 3581157 0.5 3581119 0.5

Time (Hours) Initial 4 Hrs 35 min 8 Hrs 12 Hrs 15 min 16 Hrs 20 min 20 Hrs 25 min 23 Hrs 24 Hrs 30 min 29 Hrs 33 Hrs 40 Hrs 50 min

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Time (Hours) Initial 4 Hrs 35 min 8 Hrs 12 Hrs 15 min 16 Hrs 20 min 20 Hrs 25 min 23 Hrs 24 Hrs 30 min 29 Hrs 33 Hrs 40 Hrs 50 min

Paracetamol Standard Paracetamol Test Time (Hours) Area Response % Difference Area Response % Difference Initial 9533772 NA 9674499 NA 4 Hrs 35 min 9485553 0.5 9681641 0.1 8 Hrs 9529119 0.0 9720005 0.5 12 Hrs 15 min 9562369 0.3 9713140 0.4 16 Hrs 20 min 9558274 0.3 9703185 0.3 20 Hrs 25 min 9551274 0.2 9705705 0.3 23 Hrs 9554327 0.2 9673281 0.0 24 Hrs 30 min 9551111 0.2 9659821 0.2 29 Hrs 9541020 0.1 9661378 0.1 33 Hrs 9619965 0.9 9674547 0.0 40 Hrs 50 min 9760962 2.4 9661793 0.1 Table 27: Stability of Chlorzoxazone Standard and test solution at room temperature Chlorzoxazone Standard Area Response 14101688 14074515 14105493 14182706 14175597 14185354 14165758 14150058 14064098 14290784 14499323 Chlorzoxazone Test % Difference Area Response % Difference NA 14905697 NA 0.2 14896823 0.1 0.0 15018775 0.8 0.6 14980781 0.5 0.5 14922091 0.1 0.6 14925356 0.1 0.5 14885819 0.1 0.3 14871542 0.2 0.3 14908388 0.0 1.3 14911371 0.0 2.8 14930540 0.2 BLANK CHROMATOGRAM

Table 26: Stability of Paracetamol Standard and test solution at room temperature

Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 STANDARD CHROMATOGRAM

TEST CHROMATOGRAM

LINEARITY OF DETECTOR RESPONSE OF ACECLOFENAC

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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

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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

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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

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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

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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

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Hari Kishan Reddy Ganthi et al., IJSID, 2012, 2 (4), 471-490 PHOTOLYTIC DEGRADATION SAMPLE CHROMATOGRAM HIGHER STRENGTH

stability indicating. This method has wide applicability and useful for regular quality control analysis of formulation samples. 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES (6), 13911395. Acetaminophen Drugs.com 2006, 12 (34), 250275.

Granberg RA, Rasmuson AC, Solubility of paracetamol in pure solvents, Journal of Chemical & Engineering Data, 1999, 44

The complete study results reveals that the developed and validated RP-HPLC method is accurate, precise, robust and CONCLUSION

Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S, Paracetamol: new vistas of an old drug, CNS drug reviews, mechanisms behind the acetaminophen- and NSAID-prevention of the ovarian cancer, Neoplasma, 2004, 51 (4), 239247. 648. Moller, P, Sindet-Pedersen S, Petersen C, Juhl G, Dillenschneider A, Skoglund L, Onset of acetaminophen analgesia: Cancer Research, 2008, 68 (7), 2507. (40), 1126011262.

Altinoz MA, Korkmaz R, NF-kappaB, macrophage migration inhibitory factor and cyclooxygenase-inhibitions as likely comparison of oral and intravenous routes after third molar surgery, British journal of anaesthesia, 2005, 94 (5): 642 Acetaminophen, chemicalland21.com, 2011.

Viswanathan, Feskanich, Schernhammer ES, Aspirin, NSAID and Acetaminophen use and the Risk of Endometrial Cancer, Byrant, Bronwen, Knights, Katleen, Salerno, Evelyn, Pharmacology for health professionals, Elsevier, 2007, 270. Federal Register (Notices), 2000, 65(169), 5277652777. Conference on Harmonization, Q2B: Maryland, USA, 2002), 2256. International Conference on Harmonization, Q2A: Text on Validation of Analytical Procedures, Federal Register, 1995, 60 Validation of Analytical Procedures: Methodology

10. FDA, Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation, Availability, 11. International 12. USP 25NF 20, Validation of Compendial Methods Section (1225) (United States Pharmacopeal Convention, Rockville, International Journal of Science Innovations and Discoveries, Volume 2, Issue 4, July-August 2012 Availability, Federal Register, 1997, 62 (96), 2746327467. and

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